Opsin-binding ligands, compositions and methods of use

ABSTRACT

Compounds are disclosed that are useful for treating ophthalmic conditions caused by or related to production of toxic visual cycle products that accumulate in the eye, such as dry adult macular degeneration, as well as conditions caused by or related to the misfolding of mutant opsin proteins and/or the mis-localization of opsin proteins. Compositions of these compounds alone or in combination with other therapeutic agents are also described, along with therapeutic methods of using such compounds and/or compositions. Methods of synthesizing such agents are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/361,348, having a 371(c) date of May 29, 2014, which is the U.S.National Stage of International Patent Application Serial No.PCT/US2012/066598, filed Nov. 27, 2012, which claims priority of U.S.Provisional Patent Application Ser. No. 61/565,009, filed 30 Nov., 2011,the disclosures of all of which are hereby incorporated by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 14, 2016, isnamed 246094_000063_SL.txt and is 1,147 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds and compositions thereof foruse in the treatment and/or prevention of ophthalmic diseases as well asmethods of using such compounds and/or compositions.

BACKGROUND OF THE INVENTION

A diminished visual acuity or total loss of vision may result from anumber of eye diseases or disorders caused by dysfunction of tissues orstructures in the anterior segment of the eye and/or posterior segmentof the eye. Of those that occur as a consequence of a dysfunction in theanterior segment, aberrations in the visual cycle are often involved.The visual cycle (also frequently referred to as the retinoid cycle)comprises a series of light-driven and/or enzyme catalyzed reactionswhereby a light-sensitive chromophore (called rhodopsin) is formed bycovalent bonding between the protein opsin and the retinoid agent11-cis-retinal and subsequently, upon exposure to light, the11-cis-retinal is converted to all-trans-retinal, which can then beregenerated into 11-cis-retinal to again interact with opsin. A numberof visual, ophthalmic, problems can arise due to interference with thiscycle. It is now understood that at least some of these problems are dueto improper protein folding, such as that of the protein opsin.

The main light and dark photoreceptor in the mammalian eye is the rodcell, which contains a folded membrane containing protein molecules thatcan be sensitive to light, the main one being opsin. Like other proteinspresent in mammalian cells, opsin is synthesized in the endoplasmicreticulum (i.e., on ribosomes) of the cytoplasm and then conducted tothe cell membrane of rod cells. In some cases, such as due to geneticdefects and mutation of the opsin protein, opsin can exhibit improperfolding to form a conformation that either fails to properly insert intothe membrane of the rod cell or else inserts but then fails to properlyreact with 11-cis-retinal to form native rhodopsin. In either case, theresult is moderate to severe interference with visual perception in theanimal so afflicted.

Among the diseases and conditions linked to improper opsin folding isretinitis pigmentosa (RP), a progressive ocular-neurodegenerativedisease (or group of diseases) that affects an estimated 1 to 2 millionpeople worldwide. In RP, photoreceptor cells in the retina are damagedor destroyed, leading to loss of peripheral vision (i.e., tunnel vision)and subsequent partial or near-total blindness.

In the American population the most common defect occurs as a result ofreplacement of a proline residue by a histidine residue at amino acidnumber 23 in the opsin polypeptide chain (dubbed “P23H”), caused by amutation in the gene for opsin. The result is production of adestabilized form of the protein, which is misfolded and aggregates inthe cytoplasm rather than being transported to the cell surface. Likemany other protein conformational diseases (PCDs), the clinically commonP23H opsin mutant associated with autosomal dominant RP is misfolded andretained intracellularly. The aggregation of the misfolded protein isbelieved to result in photoreceptor damage and cell death.

Recent studies have identified small molecules that stabilize misfoldedmutant proteins associated with disease. Some of these, dubbed “chemicalchaperones,” stabilize proteins non-specifically. Examples of theseinclude glycerol and trimethylamine oxide. These are not very desirablefor treating ophthalmic disease because such treatment usually requireshigh dosages that may cause toxic side effects. Other agents, dubbed“pharmacological chaperones,” (which include native ligands andsubstrate analogs) act to stabilize the protein by binding to specificsites and have been identified for many misfolded proteins, e.g.,G-protein coupled receptors. Opsin is an example of a G-protein coupledreceptor and its canonical pharmacological chaperones include the classof compounds referred to as retinoids. Thus, certain retinoid compoundshave been shown to stabilize mutant opsin proteins (see, for example,U.S. Patent Pub. 2004-0242704, as well as Noorwez et al., J. Biol.Chem., 279(16): 16278-16284 (2004)).

The visual cycle comprises a series of enzyme catalyzed reactions,usually initiated by a light impulse, whereby the visual chromophore ofrhodopsin, consisting of opsin protein bound covalently to11-cis-retinal, is converted to an all-trans-isomer that is subsequentlyreleased from the activated rhodopsin to form opsin and theall-trans-retinal product. This part of the visual cycle occurs in theouter portion of the rod cells of the retina of the eye. Subsequentparts of the cycle occur in the retinal pigmented epithelium (RPE).Components of this cycle include various enzymes, such as dehydrogenasesand isomerases, as well as transport proteins for conveying materialsbetween the RPE and the rod cells.

As a result of the visual cycle, various products are produced, calledvisual cycle products. One of these is all-trans-retinal produced in therod cells as a direct result of light impulses contacting the11-cis-retinal moiety of rhodopsin. All-trans-retinal, after releasefrom the activated rhodopsin, can be regenerated back into11-cis-retinal or can react with an additional molecule ofall-trans-retinal and a molecule of phosphatidylethanolamine to produceN-retinylidene-N-retinylethanolamine (dubbed “A2E”), an orange-emittingfluorophore that can subsequently collect in the rod cells and in theretina pigmented epithelium (RPE). As A2E builds up (as a normalconsequence of the visual cycle) it can also be converted intolipofuscin, a toxic substance that has been implicated in severalabnormalities, including ophthalmic conditions such as wet and dry agerelated macular degeneration (ARMD). A2E can also prove toxic to the RPEand has been associated with dry ARMD.

Because the build-up of toxic visual cycle products is a normal part ofthe physiological process, it is likely that all mammals, especially allhumans, possess such an accumulation to some extent throughout life.However, during surgical procedures on the eye, especially on theretina, where strong light is required over an extended period, forexample, near the end of cataract surgery and while implanting the newlens, these otherwise natural processes can cause toxicity because ofthe build-up of natural products of the visual cycle. Additionally,excessive rhodopsin activation as a result of bright light stimulationcan cause photoreceptor cell apoptosis via an AP-1 transcription factordependent mechanism. Because of this, there is a need for agents thatcan be administered prior to, during or after (or any combination ofthese) the surgical process and that has the effect of inhibitingrhodopsin activation as well as reducing the production of visual cycleproducts that would otherwise accumulate and result in toxicity to theeye, especially to the retina.

The present invention answers this need by providing small moleculeswhich noncovalently bind to opsin or mutated forms of opsin for treatingand/or amelioration such conditions, if not preventing them completely.Importantly, such agents are not natural retinoids and thus are nottightly controlled for entrance into the rod cells, where mutated formsof opsin are synthesized and/or visual cycle products otherwiseaccumulate. Therefore, such agents can essentially be titrated in asneeded for facilitating the proper folding trafficking of mutated opsinsto the cell membrane or prevention of rhodopsin activation that can leadto the excessive build-up of visual cycle products likeall-trans-retinal that in turn can lead to toxic metabolic products.Such compounds may compete with 11-cis-retinal to reduceall-trans-retinal by tying up the retinal binding pocket of opsin toprevent excessive all-trans-retinal build up. Thus, the compoundsprovided by the present invention have the advantage that they do notdirectly inhibit the enzymatic processes by which 11-cis-retinal isproduced in the eye (thus not contributing to retinal degeneration).Instead, the formation of all-trans-retinal is limited and thereby theformation of A2E is reduced. Finally, by limiting the ability of11-cis-retinal to combine with opsin to form rhodopsin, rhodopsinactivation caused by bright light stimulation especially duringophthalmic surgery is also diminished thus preventing the photocelldeath that results.

Mislocalization of photoreceptor cell visual pigment proteins (opsins)can occur in various ocular diseases, and also with normal aging. Inboth cases the accumulation of mislocalized opsin leads to the declinein viability of photoreceptor cells. With time this mislocalized opsinaccumulation leads to rod and cone cell death, retinal degeneration, andloss of vision. The present invention solves this problem by providing amethod of correcting mislocalized opsin within a photoreceptor cell bycontacting a mislocalized opsin protein with an opsin-binding agent thatbinds reversibly and/or non-covalently to said mislocalized opsinprotein, and promotes the appropriate intracellular processing andtransport of said opsin protein. This correction of mislocalizationrelieves photoreceptor cell stress, preventing decline in viability anddeath of photoreceptor cells in various diseases of vision loss, and innormal age-related decline in dim-light and peripheral rod-mediatedvision, central cone-mediated vision, and loss of night vision.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds having thestructure of Formula I, including pharmaceutically acceptable salts,solvates and hydrates thereof, and compositions of said compounds:

wherein A, R_(i), R_(j) and X—Y are as described elsewhere herein.

In a related aspect, the present invention relates to a method ofinhibiting the formation or accumulation of a visual cycle product,comprising contacting an opsin protein with a compound recited herein toinhibit formation of said visual cycle product relative to when saidcontacting does not occur.

In a further aspect, the present invention relates to a method to reducethe light toxicity associated with ophthalmic surgery by preventingrhodopsin regeneration during surgery to a mammalian eye and/or preventor slow the formation of toxic visual cycle products by fractionallypreventing rhodopsin formation during periods of light activationthereby providing a treatment of ocular conditions associated with thebuild up of visual products such as wet or dry ARMD.

In yet a further aspect, the present invention relates to a method ofcorrecting the proper folding and trafficking of mutated opsin proteins,comprising contacting a mutated opsin protein with a compound thatstabilizes the proper three dimensional conformation of the proteinrelative to when said contacting does not occur wherein the compound hasthe structure of Formula I including pharmaceutically acceptable salts,solvates and hydrates thereof.

In one embodiment, the ligand selectively binds reversibly ornon-covalently to opsin. In another embodiment, the ligand binds at ornear the 11-cis-retinal binding pocket of the opsin protein. In yetanother embodiment, the ligand binds to the opsin protein so as toinhibit or slow the covalent binding of 11-cis-retinal to the opsinprotein when the 11-cis-retinal is contacted with the opsin protein inthe presence of the ligand. In yet another embodiment, the ligand bindsto the opsin in the retinal binding pocket of opsin protein or disrupts11-cis-retinal binding to the retinal binding pocket of opsin. In yetanother embodiment, the ligand binds to the opsin protein so as toinhibit covalent binding of 11-cis-retinal to the opsin protein. In yetanother embodiment, the mammal is a human being.

In yet another embodiment, slowing or halting the progression of wet ordry ARMD is associated with reducing the level of a visual cycleproduct, for example, a visual cycle product formed fromall-trans-retinal, such as lipofuscin orN-retinylidine-N-retinylethanolamine (A2E). In yet another embodimentslowing or halting the progression of RP is associated with correctingthe folding of mutated opsins. In another embodiment, the administeringis topical administration, local administration (e.g., intraocular orperiocular injection or implant) or systemic administration (e.g., oral,injection). In yet another embodiment, the light toxicity is related toan ophthalmic procedure (e.g., ophthalmic surgery). In still anotherembodiment, the administering occurs prior to, during, or after theophthalmic surgery.

Mislocalization of photoreceptor cell visual pigment proteins (opsins)can occur in various ocular diseases, and also with normal aging. Insuch cases the accumulation of mislocalized opsin leads to the declinein viability of photoreceptor cells. With time this mislocalized opsinaccumulation leads to rod and cone cell death, retinal degeneration, andloss of vision. In one aspect, the invention provides a method ofcorrecting mislocalized opsin within a photoreceptor cell, comprisingcontacting a mislocalized opsin protein with an opsin-binding agent thatbinds reversibly and/or non-covalently to said mislocalized opsinprotein to promote the appropriate intracellular processing andtransport of said opsin protein. This correction of mislocalizationreduces photoreceptor cell stress, preventing photoreceptor cell declinein viability and death in various diseases of vision loss, and in normalage-related decline in dim-light and peripheral rod-mediated vision,central cone-mediated vision, and loss of night vision.

In various embodiments, the ocular protein mislocalization disorder isany one or more of wet or dry form of macular degeneration, retinitispigmentosa, a retinal or macular dystrophy, Stargardt's disease,Sorsby's dystrophy, autosomal dominant drusen, Best's dystrophy,peripherin mutation associate with macular dystrophy, dominant form ofStargart's disease, North Carolina macular dystrophy, light toxicity,retinitis pigmentosa, normal vision loss related aging and normal lossof night vision related to aging.

In still another embodiment, the method further involves administeringto a mammal, preferably a human being, an effective amount of at leastone additional agent selected from the group consisting of a proteasomalinhibitor, an autophagy inhibitor, a lysosomal inhibitor, an inhibitorof protein transport from the ER to the Golgi, an Hsp90 chaperoneinhibitor, a heat shock response activator, a glycosidase inhibitor, anda histone deacetylase inhibitor. In yet another embodiment, the opsinbinding ligand and the additional agent are administered simultaneously.

In still another embodiment, the opsin binding ligand and the additionalagent are each incorporated into a composition that provides for theirlong-term release. In another embodiment, the composition is part of amicrosphere, nanosphere, nano emulsion or implant. In anotherembodiment, the composition further involves administering a mineralsupplement, at least one anti-inflammatory agent, such as a steroid(e.g., any one or more of cortisone, hydrocortisone, prednisone,prednisolone, methylprednisolone, triamcinolone, betamethasone,beclamethasone and dexamethasone), or at least one anti-oxidant, such asvitamin A, vitamin C and vitamin E. In various embodiments, the opsinbinding ligand, the anti-inflammatory agent, and/or the anti-oxidant areadministered simultaneously.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof β-ionone during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

DEFINITIONS

As used throughout the disclosure, the following terms, unless otherwiseindicated, shall be understood to have the following meanings.

By “mislocalization” of a photoreceptor cell visual pigment protein (forexample, opsin, especially human opsin) is meant that the synthesizedprotein is not found at the normal or appropriate cellular location.

“Pharmacologic chaperones” refer to small molecular weight chemicalcompounds that interact with a protein (usually with a misfolded, orunfolded protein) in such a way as to alter the folding or confirmationof said protein. Such an interaction can have diverse consequences onthe cellular fate of the protein, including but not limited to leadingto increased stability and increased levels of functional protein,increased stability and increased levels of non-functional protein, ordecreased stability and decreased levels of functional or non-functionalprotein.

“Productive chaperone” refers to a pharmacologic chaperone that wheninteracting with a protein leads to an increased level of functionalprotein.

“Counterproductive, shipwreck or destructive chaperone” refers to apharmacologic chaperone that interacts with a protein (usually with amisfolded, or unfolded protein) and this interaction leads to adecreased stability and/or decreased levels of functional ornon-functional protein.

By “proteasomal inhibitor” is meant a compound that reduces aproteasomal activity, such as the degradation of a ubiquinated protein.

By “autophagy inhibitor” is meant a compound that reduces thedegradation of a cellular component by a cell in which the component islocated.

By “lysosomal inhibitor” is meant a compound that reduces theintracellular digestion of macromolecules by a lysosome. In oneembodiment, a lysosomal inhibitor decreases the proteolytic activity ofa lysosome.

By “Inhibitor of ER-Golgi protein transport” is meant a compound thatreduces the transport of a protein from the ER (endoplasmic reticulum)to the Golgi, or from the Golgi to the ER.

By “HSP90 chaperone inhibitor” is meant a compound that reduces thechaperone activity of heat shock protein 90 (HSP90). In one embodiment,the inhibitor alters protein binding to an HSP90 ATP/ADP pocket.

By “heat shock response activator” is meant a compound that increasesthe chaperone activity or expression of a heat shock pathway component.Heat shock pathway components include, but are not limited to, HSP100,HSP90, HSP70, HASP60, HSP40 and small HSP family members.

By “glycosidase inhibitor” is meant a compound that reduces the activityof an enzyme that cleaves a glycosidic bond.

By “histone deacetylase inhibitor” is meant a compound that reduces theactivity of an enzyme that deacetylates a histone.

By “reduces” or “increases” is meant a negative or positive alteration,respectively. In particular embodiments, the alteration is by at leastabout 10%, 25%, 50%, 75%, or 100% of the initial level of the proteinproduced in the absence of the opsin binding ligand.

As used herein, the term “wild-type conformation” refers to the threedimensional conformation or shape of a protein that is free of mutationsto its amino acid sequence. For opsin, this means a protein free frommutations that cause misfiling, such as the mutation designated P23H(meaning that a proline is replaced by a histidine at residue 23starting from the N-terminus). Opsin in a “wild-type conformation” iscapable of opsin biological function, including but not limited to,retinoid binding, visual cycle function, and insertion into aphotoreceptor membrane.

By “agent” is meant a small compound (also called a “compound”),polypeptide, polynucleotide, or fragment thereof. The terms compound andagent are used interchangeably unless specifically stated otherwiseherein for a particular agent or compound.

By “correcting the conformation” of a protein is meant inducing theprotein to assume a conformation having at least one biological activityassociated with a wild-type protein.

By “misfolded opsin protein” is meant a protein whose tertiary structurediffers from the conformation of a wild-type protein, such that themisfolded protein lacks one or more biological activities associatedwith the wild-type protein.

By “selectively binds” is meant a compound that recognizes and binds apolypeptide of the invention, such as opsin, but which does notsubstantially recognize and bind other molecules, especially non-opsinpolypeptides, in a sample, for example, a biological sample.

By “effective amount” or “therapeutically effective amount” is meant alevel of an agent sufficient to exert a physiological effect on a cell,tissue, or organ or a patient. As used herein, it is the amountsufficient to effect the methods of the invention to achieve the desiredresult.

By “pharmacological chaperone” is meant a molecule that upon contactinga mutant protein is able to facilitate/stabilize the proper folding ofthe protein such that it acts and functions much more like wild typeprotein than would be the case in the absence of the molecule.

By “control” is meant a reference condition. For example, where a cellcontacted with an agent of the invention is compared to a correspondingcell not contacted with the agent, the latter is the “control” or“control” cell.

By “treat” is meant decrease, suppress, attenuate, diminish, arrest, orstabilize the development or progression of a disease, preferably anocular disease, such as RP, AMD and/or light toxicity.

By “prevent” is meant reduce the risk that a subject will develop acondition, disease, or disorder, preferably an ocular disease, such asRP, AMD and/or light toxicity.

By “competes for binding” is meant that a compound of the invention andan endogenous ligand are incapable of binding to a target at the sametime. Assays to measure competitive binding are known in the art, andinclude, measuring a dose dependent inhibition in binding of a compoundof the invention and an endogenous ligand by measuring t_(1/2), forexample.

A “pharmaceutically acceptable salt” is a salt formed from an acid or abasic group of one of the compounds of the invention. Illustrative saltsinclude, but are not limited to, sulfate, citrate, acetate, oxalate,chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesuifonate, and pamoate (i.e.,1,1′-methytene-bis-(2-hydroxy-3-naphthoate)) salts.

The term “pharmaceutically acceptable salt” also refers to a saltprepared from a compound of the invention having an acidic functionalgroup, such as a carboxylic acid functional group, and apharmaceutically acceptable inorganic or organic base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine;tributyl amine; pyridine; N-methyl-N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), suchas mono-, bis-, or tris-(2-hydroxyethyl)-amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)-amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

The term “pharmaceutically acceptable salt” also refers to a saltprepared from a compound disclosed herein, e.g., a salt of a compound ofExample 1, having a basic functional group, such as an amino functionalgroup, and a pharmaceutically acceptable inorganic or organic acid.Suitable acids include, but are not limited to, hydrogen sulfate, citricacid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide,hydrogen iodide, nitric acid, phosphoric acid, isonicotinic acid, lacticacid, salicylic acid, tartaric acid, ascorbic acid, succinic acid,maleic acid, besylic acid, fumaric acid, gluconic acid, glucaronic acid,saccharic acid, formic acid, benzoic acid, glutamic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, andp-toluenesulfonic acid.

The term “pharmaceutically-acceptable excipient” as used herein meansone or more compatible solid or liquid tiller, diluents or encapsulatingsubstances that are suitable for administration into a human. The term“excipient” includes an inert substance added to a pharmacologicalcomposition to further facilitate administration of a compound. Examplesof excipients include but are not limited to calcium carbonate, calciumphosphate, various sugars and types of starch, cellulose derivatives,gelatin, vegetable oils and polyethylene glycols.

The term “carrier” denotes an organic or inorganic ingredient, naturalor synthetic, with which the active ingredient is combined to facilitateadministration.

The term “parenteral” includes subcutaneous, intrathecal, intravenous,intramuscular, intraperitoneal, or infusion.

The term “visual cycle product” refers to a chemical entity produced asa natural product of one or more reactions of the visual cycle (thereactive cycle whereby opsin protein binds 11-cis-retinal to formrhodopsin, which accepts a light impulse to convert 11-cis-retinal toall trans-retinal, which is then released from the molecule toregenerate opsin protein with subsequent binding of a new 11-cis-retinalto regenerate rhodopsin). Such visual cycle products include, but arenot limited to, all-trans-retinal, lipofuscin and A2E.

The term “light toxicity” refers to any condition affecting vision thatis associated with, related to, or caused by the production and/oraccumulation of visual cycle products. Visual cycle products include,but are not limited to, all-trans-retinal, lipofuscin or A2E. In oneparticular embodiment, light toxicity is related to exposure of the eyeto large amounts of light or to very high light intensity, occurring,for example, during a surgical procedure on the retina.

The term “opsin” refers to an opsin protein, preferably a mammalianopsin protein, most preferably a human opsin protein. In one embodiment,the opsin protein is in the wild-type (i.e., physiologically active)conformation. One method of assaying for physiological activity isassaying the ability of opsin to bind 11-cis-retinal and form activerhodopsin. A mutant opsin, such as the P23H mutant, that is ordinarilymisfolded has a reduced ability to bind 11-cis-retinal, and thereforeforms little or no rhodopsin. Where the conformation of the mutant opsinhas been corrected (for example, by binding to a pharmacologicalchaperone), the opsin is correctly inserted into the rod cell membraneso that its conformation is the same, or substantially the same, as thatof a non-mutant opsin. This allows the mutant opsin to bind11-cis-retinal to form active rhodopsin. Therefore, the methods of theinvention operate to reduce the formation of visual cycle products.

“Alkyl” refers to an unbroken non-cyclic chain of carbon atoms that maybe substituted with other chemical groups. It may also be branched orunbranched, substituted or unsubstituted.

“Lower alkyl” refers to a branched or straight chain acyclic alkyl groupcomprising one to ten carbon atoms, preferably one to eight carbonatoms, more preferably one to six carbon atoms. Exemplary lower alkylgroups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, pentyl, neopentyl, iso-amyl, hexyl, and octyl.

“Hydroxy” refers to —OH. “Oxy” refers to —O—. “Oxo” refers to ═O.

“Organic acid” refers to compound having at least one carbon atom andone or more functional groups capable of releasing a proton to a basicgroup. The organic acid preferably contains a carboxyl, a sulfonic acidor a phosphoric acid moiety. Exemplary organic acids include aceticacid, benzoic acid, citric acid, camphorsulfonic acid, methanesulfonicacid, taurocholic acid, chlordronic acid, glyphosphate and medronicacid.

“Inorganic acid” refers to a compound that does not contain at least onecarbon atom and is capable of releasing a proton to a basic group.Exemplary inorganic acids include hydrochloric acid, sulfuric acid,nitric acid and phosphoric acid.

“Organic base” refers to a carbon containing compound having one or morefunctional groups capable of accepting a proton from an acid group. Theorganic base preferably contains an amine group. Exemplary organic basesinclude triethylamine, benzyldiethylamine, dimethylethyl amine,imidazole, pyridine and piperidine.

“Independently selected” groups are groups present in the same structurethat need not all represent the same substitution. For example, wheretwo substituents are represented as NOR_(A) and each R_(A) is said to beindependently selected from H, methyl, ethyl, etc., this means thatwhere one R_(A) is methyl, the other R_(A) may be methyl but could be Hor ethyl (or any other recited substitution).

Some of the compounds for use in the methods of the present inventionmay contain one or more chiral centers and therefore may exist inenantiomeric and diastereomeric forms. The scope of the presentinvention is intended to cover use of all isomers per se, as well asmixtures of cis and trans isomers, mixtures of diastereomers and racemicmixtures of enantiomers (optical isomers) as well. Further, it ispossible using well known techniques to separate the various forms, andsome embodiments of the invention may feature purified or enrichedspecies of a given enantiomer or diastereomer.

A “pharmacological composition” refers to a mixture of one or more ofthe compounds described herein, or pharmaceutically acceptable saltsthereof, with other chemical components, such as pharmaceuticallyacceptable carriers and/or excipients. The purpose of a pharmacologicalcomposition is to facilitate administration of a compound to anorganism.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agent fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. A physiologically acceptablecarrier should not cause significant irritation to an organism and doesnot abrogate the biological activity and properties of the administeredcompound.

A “solvate” is a complex formed by the combination of a solute (e.g., ametalloprotease inhibitor) and a solvent (e.g., water). See J. Honig etal., The Van Nostrand Chemist's Dictionary, p. 650 (1953).

The terms “optical isomer”, “geometric isomer” (e.g., a cis and/or transisomer), “stereoisomer”, and “diastereomer” have the accepted meanings(see, e.g., Hawley's Condensed Chemical Dictionary, 11th Ed.). Theillustration of specific protected forms and other derivatives of thecompounds of the instant invention is not intended to be limiting. Theapplication of other useful protecting groups, salt forms, prodrugsetc., is within the ability of the skilled artisan.

A “prodrug” is a form of a drug that must undergo chemical conversion bymetabolic processes before becoming an active, or fully active,pharmacological agent. A prodrug is not active, or is less active, inits ingested or absorbed or otherwise administered form. For example, aprodrug may be broken down by bacteria in the digestive system intoproducts, at least one of which will become active as a drug.Alternatively, it may be administered systemically, such as byintravenous injection, and subsequently be metabolized into one or moreactive molecules.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, small molecule ligands arecapable of reversibly binding non-covalently to the opsin protein andinhibiting the binding of 11-cis-retinal, to an opsin retinal bindingpocket. Such interference with retinal binding reduces the formation ofvisual cycle products, such as all-trans-retinal, and thereby inhibitsthe production of compounds such as lipofuscin and A2E with resultingreduced risk and occurrence of toxicity that can result fromaccumulation of these substances. Such compounds, acting aspharmacologic chaperones, are also able to facilitate the proper foldingand trafficking of mutant opsins associated with RP. Additionally, byinhibiting 11-cis-retinal binding and rhodopsin formation, the excessivestimulation and resulting activation of rhodopsin caused by exposure ofthe retina to bright light especially during retinal surgery reducesphotocell death.

Certain synthetic retinoids (compounds structurally related to retinol(Vitamin A alcohol)) have been reported to bind to opsin. In theembodiments of the present invention, non-retinoid small molecules(compounds having a molecular weight less than about 1000 daltons, lessthan 800, less than 600, less than 500, less than 400, or less thanabout 300 daltons) have been found to bind to opsin.

The invention features compositions and methods that are useful forreducing formation of visual cycle products and toxicity associated withthe accumulation of such products in vivo, reducing the probability ofapoptotic events associated with excessive rhodopsin activation as wellas preventing rod cell death due to aberrant processing and traffickingof mutant opsin proteins associated with RP.

Mislocalization of photoreceptor cell visual pigment proteins (opsins)can occur in various ocular diseases, and also with normal aging. Insuch cases the accumulation of mislocalized opsin leads to the declinein viability of photoreceptor cells. With time this mislocalized opsinaccumulation leads to rod and cone cell death, retinal degeneration, andloss of vision.

In one aspect, the invention provides a method of correctingmislocalized opsin within a photoreceptor cell, comprising contacting amislocalized opsin protein with an opsin-binding agent that bindsreversibly and/or non-covalently to said mislocalized opsin protein,thereby promoting correct intracellular processing and transport of saidopsin protein. Such opsin-binding agent is referred to as a “ProductiveChaperone.”

Such correction of mislocalization reduces photoreceptor cell stress,preventing photoreceptor cell decline in viability and death in variousdiseases of vision loss, and in normal age-related decline in dim-lightand peripheral rod-mediated vision, central cone-mediated vision, andloss of night vision.

In another aspect of the invention, the opsin-binding agent promotes thedegradation of the mislocalized opsin protein. This type ofopsin-binding agent is referred to as a “Counterproductive”, Shipwreck“,or “Destructive Chaperone.”

Enhancing the degradation of the mislocalized opsin by such an agentreduces the amount of mislocalized protein, thereby relievingphotoreceptor cell stress, preventing decline in viability and death ofphotoreceptor cells in diseases of vision loss, as well as in normalage-related decline in dim-light and peripheral rod-mediated vision,central cone-mediated vision, and loss of night vision.

In embodiments of the foregoing, the ocular protein mislocalizationdisorder is one or more of wet or dry form of macular degeneration,retinitis pigmentosa, a retinal or macular dystrophy, Stargardt'sdisease, Sorsby's dystrophy, autosomal dominant drusen, Best'sdystrophy, peripherin mutation associate with macular dystrophy,dominant form of Stargart's disease, North Carolina macular dystrophy,light toxicity, retinitis pigmentosa, normal vision loss related agingand normal loss of night vision related to aging.

Opsin, the GPCR (G-protein coupled receptor) responsible for vision,readily regenerates with 11-cis-retinal to form the visual pigmentrhodopsin. The pigment is generated by formation of a protonated Schiffbase between the aldehyde group of 11-cis-retinal and the E-amino groupof L-lysine in opsin (Matsumoto and Yoshizawa, Nature 1975 Dec. 11;258(5535):523-6).

Thus; the present invention provides compositions and methods of use ofsmall molecule compounds that bind to wild type and mutant opsins andcompete with, or other wise prevent, 11-cis-retinal from combining withopsin to form rhodopsin and thereby inhibit formation of 11-cis-retinaland other visual cycle products.

In one embodiment, the invention provides opsin binding ligands ofFormula I and pharmaceutically acceptable salts thereof:

wherein A is:

R¹ and R² are independently:

-   -   1) hydrogen,    -   2) —CH₃, or    -   3) —CH₂CH₃;

R³ is:

-   -   1) hydrogen,    -   2) —CH₃,    -   3) —CH₂CH₃, or    -   4) deuteron;

R⁴ is:

-   -   1) hydrogen,    -   2) —CH₃, or    -   3) deuteron;

R_(a) and R_(b) are each independently:

-   -   1) hydrogen, or    -   2) —CH₃;

T is:

-   -   1) CH₂,    -   2) CH₂CH₂, or    -   3) absent;

R_(i), and R_(j) are each independently:

-   -   1) hydrogen,    -   2) hydroxyl, or    -   3) lower alkyl;

R_(i) and R_(j) taken together are oxo (═O);

X—Y is:

-   -   1) —N(CONH₂)—CH₂—, or    -   2) —CH₂—N(CONH₂)—;

In preferred embodiments, the compound has the structure of Formula Iwherein R_(i) is hydroxy and R_(j) is hydrogen or lower alkyl andwherein R¹ and R² are each independently methyl or ethyl, R_(a) andR_(b) are each selected from hydrogen or methyl, more preferably whereinR_(i) is hydroxy and R_(j) is hydrogen or methyl, both of R¹ and R² aremethyl and both R_(a) and R_(b) are hydrogen and R³ is hydrogen ormethyl, and most preferably wherein R¹, R² and R³ are each methyl, R_(i)is hydroxy, R_(j) is hydrogen or methyl and both R_(a) and R_(b) ishydrogen and X—Y is —N(CONH₂)—CH₂—.

In specific embodiments the opsin binding compound of Formula I is(wherein each compound number corresponds to the number of the examplewhere it was prepared):

-   6-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 1);-   6-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinone-2(1H)-carboxamide    (Compound 2);-   6-((S)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 3);-   6-((1R,6S)-2,2,6-trimethylcyclohexanecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 4);-   6-((R)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 5);-   7-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 6);-   7-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 7);-   6-(2,5,5-trimethylcyclopent-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 8);-   6-(hydroxy(2,5,5-trimethylcyclopent-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 9);-   6-(3,3,6,6-tetramethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 10);-   6-(7,7-dimethylcyclohept-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 11);-   6-(7,7-dimethylcyclohept-1-enyl)(hydroxy)methyl)-3,4-dihydroisoqunoline-2(1H)-carboxamide    (Compound 12);-   6-((R)-1-hydroxy-1-((1R,6S)-2,2,6-trimethylcyclohexyl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 13);-   6-(hydroxy(3,3,6,6-tetramethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 14)    including all pharmaceutically acceptable salts, hydrates, or    solvates thereof.

All compound names were derived using ChemBioDraw 11.0.1 and thestereochemistry of new chiral centers of products resulting from theaddition to chiral aldehydes or ketones was assigned based upon CramsRule of asymmetric induction (Cram and Elhafez, J. Am. Chem. Soc.,74:5828-5835 (1952)).

Especially preferred examples of the compounds of the invention, andmethods using said compounds, include compounds selected from one ormore of the group consisting of compounds 2, 3 and 5 including allpharmaceutically acceptable salts, solvates and hydrates thereof.

The present invention also provides thereapeutic compositions,comprising a therapeutically effective amount of a compound of Formula I

wherein A is:

R¹ and R² are independently:

-   -   1) hydrogen,    -   2) —CH₃, or    -   3) —CH₂CH₃;

R³ is:

-   -   1) hydrogen,    -   2) —CH₃,    -   3) —CH₂CH₃, or    -   4) deuteron;

R⁴ is:

-   -   1) hydrogen,    -   2) —CH₃, or    -   3) deuteron;

R_(a) and R_(b) are each independently:

-   -   1) hydrogen, or    -   2) —CH₃;

T is:

-   -   1) CH₂,    -   2) CH₂CH₂, or    -   3) absent;

R_(i) and R_(j) are each independently:

-   -   1) hydrogen,    -   2) hydroxyl, or    -   3) lower alkyl;

R_(i) and R_(j) when taken together are ═O;

X—Y is:

-   -   1) —N(CONH₂)—CH₂—, or    -   2) —CH₂—N(CONH₂)—,

including pharmaceutically acceptable salts, solvates and hydratesthereof.

In preferred embodiments, the compounds and compositions of theinvention include embodiments of Formula I wherein R_(i) is hydroxy andR_(j) is hydrogen or lower alkyl, wherein R¹ and R² are eachindependently methyl or ethyl, wherein R_(a) and R_(b) are hydrogen ormethyl, and wherein R¹ and R² are each independently methyl or ethyl, orcombinations of these. In one such preferred embodiment, R_(i) ishydroxy and R_(j) is hydrogen or lower alkyl, R¹ and R² are eachindependently methyl or ethyl, and R_(a) and R_(b) are hydrogen ormethyl.

In preferred embodiments, the compounds and compositions of theinvention include embodiments of Formula I wherein R_(i) is hydroxy andR_(j) is hydrogen or methyl, wherein each of R¹ and R² is methyl,wherein R_(a) and R_(b) are each hydrogen, wherein R³ is hydrogen ormethyl, or combinations of these. In one such preferred embodiment,R_(i) is hydroxy and R_(j) is hydrogen or methyl, each of R¹ and R² ismethyl, R_(a) and R_(b) are each hydrogen and R³ is hydrogen or methyl.

In preferred embodiments, the compounds and compositions of theinvention include embodiments of Formula I wherein R¹, R² and R³ areeach methyl, wherein R_(i) is hydroxy and R_(j) is hydrogen or methyl,wherein each of R_(a) and R_(b) is hydrogen, wherein X—Y is—N(CONH₂)—CH₂—. In one such preferred embodiment, R¹, R² and R³ methyl,R_(i) is hydroxy and R_(j) is hydrogen or methyl and both R_(a) andR_(b) is hydrogen and X—Y is —N(CONH₂)—CH₂—.

In another preferred embodiment, the invention provides a compositioncomprising a therapeutically effective amount of a compound selectedfrom the group consisting of

-   6-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 1);-   6-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 2);-   6-((S)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 3);-   6-((1R,6S)-2,2,6-trimethylcyclohexanecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 4);-   6-((R)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 5);-   7-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 6);-   7-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 7);-   6-(2,5,5-trimethylcyclopent-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 8);-   6-(hydroxy(2,5,5-trimethylcyclopent-1-enyl)methyl)-3,4-dihydrosoquinoline-2(1H)-carboxamide    (Compound 9);-   6-(3,3,6,6-tetramethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 10);-   6-(7,7-dimethylcyclohept-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 11);-   6-(7,7-dimethylcyclohept-1-enyl)(hydroxy)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 12);-   6-((R)-1-hydroxy-1-((1R,6S)-2,2,6-trimethylcyclohexyl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound13);-   6-(hydroxy(3,3,6,6-tetramethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound14)    including all pharmaceutically acceptable salts, hydrates, or    solvates thereof.

In an much preferred embodiment, the invention provides a compositioncomprising a therapeutically effective amount of a compound selectedfrom the group consisting of compounds 2, 3 and 5 including allpharmaceutically acceptable salts, solvates and hydrates thereof.

Another embodiment of the invention provides the opsin binding ligandmetabolites of the opsin binding compounds. These metabolites, includebut are not limited to, degradation products, hydrolysis products,gluconoride adducts and the like, of the opsin binding compounds andpharmaceutically acceptable salts thereof, of the opsin compounds.

Another embodiment of the invention provides processes for making thenovel compounds of the invention and to the intermediates useful in suchprocesses. The reactions are performed in solvents appropriate to thereagents and materials used are suitable for the transformations beingeffected. It is understood by one skilled in the art of organicsynthesis that the functionality present in the molecule must beconsistent with the chemical transformation proposed. This will, onoccasion, necessitate judgment by the routineer as to the order ofsynthetic steps, protecting groups required, and deprotectionconditions. Substituents on the starting materials may be incompatiblewith some of the reaction conditions required in some of the methodsdescribed, but alternative methods and substituents compatible with thereaction conditions will be readily apparent to one skilled in the art.The use of sulfur, nitrogen and oxygen protecting groups is well knownfor protecting thiol, amino and alcohol groups against undesirablereactions during a synthetic procedure and many such protecting groupsare known and described by, for example, Greene and Wuts, ProtectiveGroups in Organic Synthesis, Third Edition, John Wiley & Sons, New York(1999).

Compounds of the invention that have one or more asymmetric carbon atomsmay exist as the optically pure enantiomers, pure diastereomers,mixtures of enantiomers, mixtures of diastereomers, racemic mixtures ofenantiomers, diasteromeric racemates or mixtures of diastereomericracemates. It is to be understood that the invention anticipates andincludes within its scope all such isomers and mixtures thereof.

The chemical reactions described herein are generally disclosed in termsof their broadest application to the preparation of the compounds ofthis invention. Occasionally, the reactions may not be applicable asdescribed to each compound included within the disclosed scope. Thecompounds for which this occurs will be readily, recognized by oneskilled in the art. In all such cases, either the reactions can besuccessfully performed by conventional modifications known to oneskilled in the art, e.g., by appropriate protection of interferinggroups, by changing to alternative conventional reagents, by routinemodification of reaction conditions, or other reactions disclosed hereinor otherwise conventional, will be applicable to the preparation of thecorresponding compounds of this invention. In all preparative methods,all starting materials are known or readily prepared from known startingmaterials.

Methods of the Invention

The present invention provides a method of using compounds of theFormula I for reducing the formation of toxic visual cycle products,comprising contacting an opsin protein with small molecule ligands thatreversibly bind to said opsin protein to inhibit 11-cis-retinal bindingin said binding pocket, thereby reducing formation of toxic visual cycleproducts associated with wet or dry ARMD and reducing photocellapoptosis associated with excessive rhodopsin activation as a result ofbright light stimulation.

The present invention also provides a method of use of compounds of theFormula I for treating, preventing or reducing the risk of lighttoxicity in a mammal, comprising administering to a mammal, at risk ofdeveloping an ophthalmic condition that is related to the formation oraccumulation of a visual cycle product or apoptotic photocell death.

The present invention also provides a method of use of compounds of theFormula I for treating, preventing or reducing the risk of lighttoxicity in a mammal, comprising administering to a mammal, at risk ofdeveloping an ophthalmic condition that is related to the formation oraccumulation of a visual cycle product or apoptotic photocell death, aneffective amount of a that small molecule ligand that reversibly binds(for example, at or near the retinal binding pocket) to an opsin proteinpresent in the eye of said mammal, for example, to inhibit11-cis-retinal binding in said binding pocket, thereby reducing lighttoxicity and photocell apoptosis.

The present invention also provides a method of use of compounds of theFormula I for treating, preventing or reducing the risk of RP in amammal, comprising administering to a mammal, at risk of RP related tothe improper folding and trafficking of mutant opsins, an effectiveamount of a that small molecule ligand that reversibly binds (forexample, at or near the retinal binding pocket) to an opsin proteinpresent in the eye of said mammal, for example, to inhibit11-cis-retinal binding in said binding pocket, thereby reducing thevision loss caused by RP.

In specific examples of such methods, the small molecule ligand isselective for binding to opsin and/or the small molecule ligand binds tosaid opsin in the retinal binding pocket of said opsin protein and/orthe small molecule ligand binds to said opsin protein so as to inhibitcovalent binding of 11-cis-retinal to said opsin protein when said11-cis-retinal is contacted with said opsin protein when said smallmolecule ligand is present and/or the mammal is a human being.

In one embodiment, the invention provides a method of inhibiting theformation or accumulation of a visual cycle product, comprisingcontacting an opsin protein with a compound of Formula I

wherein A is:

R¹ and R² are independently:

-   -   1) hydrogen,    -   2) —CH₃, or    -   3) —CH₂CH₃;

R³ is:

-   -   1) hydrogen,    -   2) —CH₃,    -   3) —CH₂CH₃, or    -   4) deuteron;

R⁴ is:

-   -   1) hydrogen,    -   2) —CH₃, or    -   3) deuteron;

R_(a) and R_(b) are each independently:

-   -   1) hydrogen, or    -   2) —CH₃;

T is:

-   -   1) CH₂,    -   2) CH₂CH₂, or    -   3) absent;

R_(i) and R_(j) are each independently:

-   -   1) hydrogen,    -   2) hydroxyl, or    -   3) lower alkyl;

R_(i) and R_(j) when taken together are ═O;

X—Y is:

-   -   1) —N(CONH₂)—CH₂—, or    -   2) —CH₂—N(CONH₂)—;

including pharmaceutically acceptable salts, solvates and hydratesthereof.

In preferred embodiments of this method, the visual cycle product is atoxic visual cycle product, for example, wherein said toxic visual cycleproduct is lipofuscin or N-retinylidene-N-retinylethanolamine (A2E).

In another embodiment, the invention provides a method of treating orpreventing an ophthalmic condition in a subject at risk thereof,comprising administering to the subject an effective amount of acompound of Formula I, with the same limitations on constituent groupsas stated above and elsewhere herein for Formula 1 and including allpharmaceutically acceptable salts, solvates and hydrates thereof.

Preferred embodiments of the above include methods wherein the compoundreduces mislocalization of said opsin protein, such as wherein saidcompound binds to said opsin protein by hydrogen bonding. Otherpreferred embodiment include those wherein said opsin protein is presentin a cell, especially a cone cell or rod cell, such as wherein said cellis present in a mammalian eye.

In other preferred embodiments of the above method, the ophthalmiccondition is an ocular protein mislocalization disorder, such as oneselected from the group consisting of wet or dry age related maculardegeneration (ARMD), retinitis pigmentosa (RP), a retinal or maculardystrophy, Stargardt's disease, Sorsby's dystrophy, autosomal dominantdrusen, Best's dystrophy, peripherin mutation associate with maculardystrophy, dominant form of

Stargart's disease, North Carolina macular dystrophy, light toxicity,normal vision loss related aging and normal loss of night vision relatedto aging.

In a most preferred embodiment, the ophthalmic condition is retinitispigmentosa (RP), especially where said RP is caused by aberrantopsin-folding.

In preferred embodiments of the methods of the invention, the compoundsand compositions used in the methods of the invention includeembodiments of Formula I wherein R_(i) is hydroxy and R_(j) is hydrogenor lower alkyl, wherein R¹ and R² are each independently methyl orethyl, wherein R_(a) and R_(b) are hydrogen or methyl, or combinationsof these. In one such preferred embodiment, R_(i) is hydroxy and R_(j)is hydrogen or lower alkyl, R¹ and R² are each independently methyl orethyl, and R_(a) and R_(b) are hydrogen or methyl.

In preferred embodiments of the methods of the invention, the compoundsand compositions used in the methods of the invention includeembodiments of Formula I wherein R_(i) is hydroxy and R_(j) is hydrogenor methyl, wherein each of R¹ and R² is methyl, wherein R_(a) and R_(b)are each hydrogen, wherein R³ is hydrogen or methyl, or combinations ofthese. In one such preferred embodiment, R_(i) is hydroxy and R_(j) ishydrogen or methyl, each of R¹ and R² is methyl, R_(a) and R_(b) areeach hydrogen and R³ is hydrogen or methyl.

In preferred embodiments of the methods of the invention, the compoundsand compositions used in the methods of the invention includeembodiments of Formula I wherein R¹, R² and R³ are each methyl, whereinR_(i) is hydroxy and R_(j) is hydrogen or methyl, wherein each of R _(a)and R_(b) is hydrogen, wherein X—Y is —N(CONH₂)—CH₂—. In one suchpreferred embodiment, R¹, R² and R³ methyl, R_(i) is hydroxy and R_(j)is hydrogen or methyl and both R_(a) and R_(b) is hydrogen and X—Y is—N(CONH₂)—CH₂—.

In one preferred embodiment, the invention provides a method ofinhibiting the formation or accumulation of a visual cycle product,comprising contacting an opsin protein with a compound selected from thegroup consisting of

-   6-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 1);-   6-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 2);-   6-((S)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 3);-   6-((1R,6S)-2,2,6-trimethylcyclohexanecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 4);-   6-((R)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 5);-   7-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 6);-   7-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 7);-   6-(2,5,5-trimethylcyclopent-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 8);-   6-(hydroxy(2,5,5-trimethylcyclopent-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 9);-   6-(3,3,6,6-tetramethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 10);-   6-(7,7-dimethylcyclohept-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 11);-   6-((7,7-dimethylcyclohept-1-enyl)(hydroxy)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 12);-   6-((R)-1-hydroxy-1-((1R,6S)-2,2,6-trimethylcyclohexyl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound13);-   6-(hydroxy(3,3,6,6-tetramethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound14)

including all pharmaceutically acceptable salts, hydrates, or solvatesthereof.

In another preferred embodiment, the invention provides a method oftreating or preventing an ophthalmic condition in a subject at riskthereof or afflicted therewith, comprising administering to the subjectan effective amount of a compound selected from the group consisting of:

-   6-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 1);-   6-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 2);-   6-((S)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 3);-   6-((1R,6S)-2,2,6-trimethylcyclohexanecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 4);-   6-((R)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 5);-   7-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 6);-   7-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 7);-   6-(2,5,5-trimethylcyclopent-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 8);-   6-(hydroxy(2,5,5-trimethylcyclopent-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 9);-   6-(3,3,6,6-tetramethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 10);-   6-(7,7-dimethylcyclohept-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 11);-   6-(7,7-dimethylcyclohept-1-enyl)(hydroxy)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 12);-   6-(R)-1-hydroxy-1-((1R,6S)-2,2,6-trimethylcyclohexyl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 13);-   6-(hydroxy(3,3,6,6-tetramethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide    (Compound 14)

including all pharmaceutically acceptable salts, hydrates, or solvatesthereof.

In a most preferred embodiment, the invention provides a method ofinhibiting the formation or accumulation of a visual cycle product,comprising contacting an opsin protein with a compound selected from thegroup consisting of compounds 2, 3 and 5 including all pharmaceuticallyacceptable salts, solvates and hydrates thereof.

In another especially preferred embodiment, the invention provides amethod of treating or preventing an ophthalmic condition in a subject atrisk thereof or afflicted therewith, comprising administering to thesubject an effective amount of a compound selected from the groupconsisting of compounds 2, 3 and 5 including all pharmaceuticallyacceptable salts, solvates and hydrates thereof.

In accordance with the above methods, light toxicity is related to anophthalmic procedure, for example, ophthalmic surgery. Said agent may beadministered prior to, during or after said surgery (or at any one ormore of those times).

In specific embodiments of the methods of the invention, the nativeopsin protein is present in a cell, such as a rod cell, preferably, amammalian and more preferably a human cell. In specific embodiments, thesmall molecule ligands of the invention inhibit binding of11-cis-retinal in the binding pocket of opsin and slow the visual cyclethereby reducing the formation of all-trans-retinal, or a toxic visualcycle product formed from it, such as lipofuscin orN-retinylidene-N-retinylethanolamine (A2E). Alternatively, photocellapoptosis as a result of excessive rhodopsin activation is reduced orprevented by inhibition of rhodopsin formation. Additionally, improperfolding and trafficking of mutant opsin proteins associated with RP isreduced.

In methods of the invention, administering is preferably by topicaladministration (such as with an eye wash) or by systemic administration(including oral, intraocular injection or periocular injection). By wayof preferred example, the ophthalmic condition to be treated is lighttoxicity, such as that resulting from ocular surgery, for example,retinal or cataract surgery.

Also encompassed is an ophthalmologic composition comprising aneffective amount of compounds of the Formula I in a pharmaceuticallyacceptable carrier, wherein said agent reversibly binds non-covalently(for example, at or near the retinal binding pocket) to said opsinprotein to inhibit 11-cis-retinal binding in said pocket, preferablywhere the small molecule ligand is selective for opsin protein.

The present invention further provides a screening method foridentifying a small molecule ligand that reduces light toxicity in amammalian eye, comprising:

(a) contacting a native opsin-protein with a test compound in thepresence of 11-cis-retinal and under conditions that promote the bindingof the test compound and the 11-cis-retinal to the native opsin protein;and

(b) determining a reversible reduction in rate of formation of rhodopsinrelative to the rate when said test compound is not present,

thereby identifying said test compound as a small molecule ligand thatreduces light toxicity in a mammalian eye. In a preferred embodiment,said test compound is structurally related to a compound disclosedherein.

The compounds of the Formula I may be administered along with otheragents, including a mineral supplement, an anti-inflammatory agent, suchas a steroid, for example, a corticosteroid, and/or an anti-oxidant.Among the corticosteroids useful for such administration are thoseselected from the group consisting of cortisone, hydrocortisone,prednisone, prednisolone, methylprednisolone, triamcinolone,betamethasone, beclamethasone and dexamethasone. Useful anti-oxidantsinclude vitamin A, vitamin C and vitamin E.

The methods of the invention also contemplate reducing light toxicity byusing at least one additional agent (in addition to the compounds of theFormula I selected from the group consisting of a proteasomal inhibitor,an autophagy inhibitor, a lysosomal inhibitor, an inhibitor of proteintransport from the ER to the Golgi, an Hsp90 chaperone inhibitor, a heatshock response activator, a glycosidase inhibitor, and a histonedeacetylase inhibitor, wherein the small molecule opsin binding and theadditional compound are administered simultaneously or within fourteendays of each other in amounts sufficient to treat the subject.

In a particular example of the methods of the invention, the compoundsof the Formula I and the additional compound are administered within tendays of each other, within five days of each other, within twenty-fourhours of each other and preferably are administered simultaneously. Inone example, the small molecule opsin binding and the additionalcompound are administered directly to the eye. Such administration maybe intraocular or intravitrial. In other examples, the small moleculeopsin binding and the additional compound are each incorporated into acomposition that provides for their long-term release, such as where thecomposition is part of a microsphere, nanosphere, nano emulsion orimplant.

As described herein, the compounds of the Formula I are useful in themethods of the invention are available for use alone or in combinationwith one or more additional compounds to treat or prevent conditionsassociated with excessive rhodopsin activation, such as light toxicity,for example, resulting from ocular surgical procedures. In oneembodiment, compounds of the Formula I of the invention is administeredwithout an additional active compound. In another embodiment, compoundsof the Formula I of the invention is used in combination and withanother active compound (e.g., as discussed herein). In still anotherexemplary embodiment, compounds of the Formula I are administered incombination with the proteasomal inhibitor MG132, the autophagyinhibitor 3-methyladenine, a lysosomal inhibitor ammonium chloride, theER-Golgi transport inhibitor brefeldin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and the histone deacetylase inhibitor Scriptaid, can be usedto reduce formation of visual cycle products and cell apoptosis as aresult of excessive rhodopsin activation.

As described herein, the compounds of the Formula I are useful in themethods of the invention are available for use alone or in combinationwith one or more additional compounds to treat or prevent the aberrantprocessing and trafficking of mutant opsin proteins associated with rodcell death as a result of RP. In one embodiment, compounds of theFormula I of the invention is administered without an additional activecompound. In another embodiment, compounds of the Formula I of theinvention is used in combination and with another active compound (e.g.,as discussed herein). In still another exemplary embodiment, compoundsof the Formula I are administered in combination with the proteasomalinhibitor MG132, the autophagy inhibitor 3-methyladenine, a lysosomalinhibitor ammonium chloride, the ER-Golgi transport inhibitor brefeldinA, the Hsp90 chaperone inhibitor Geldamycin, the heat shock responseactivator Celastrol, the glycosidase inhibitor, and the histonedeacetylase inhibitor Scriptaid, can be used to reduce or prevent therod cell death and resulting blindness associated with RP.

As described herein, the compounds of the Formula I are useful in themethods of the invention are available for use alone or in combinationwith one or more additional compounds to treat or prevent conditionsassociated with production and accumulation of toxic visual cycleproducts derived from all-trans-retinal, such as lipofucin and A2E, forexample, the blindness associated with wet or dry ARMD. In oneembodiment, compounds of the Formula I of the invention are administeredwithout an additional active compound. In another embodiment, compoundsof the Formula I of the invention are used in combination and withanother active compound (e.g., as discussed herein). In still anotherexemplary embodiment, compounds of the Formula I are administered incombination with the proteasomal inhibitor MG132, the autophagyinhibitor 3-methyladenine, a lysosomal inhibitor ammonium chloride, theER-Golgi transport inhibitor brefeldin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and the histone deacetylase inhibitor Scriptaid, can be usedto reduce formation of toxic visual cycle product metabolites and photocell death as a result of dry ARMD.

In a typical competition assay of the invention, a compound is soughtthat will tie up the retinal binding pocket of the opsin protein. Thus,the assay seeks to identify a small molecule opsin binding compound (onethat will not be tightly regulated by the retina as to amount enteringrod cells) that competes with or prevents 11-cis-retinal or9-cis-retinal from forming rhodopsin or isorhodopsin. Over time, thiswill slow the rate of formation of rhodopsin relative to the rate when11-cis-retinal alone is present. in one embodiment, the assay isconducted in the presence of 11-cis-retinal, and the rate of formationof rhodopsin is measured as a way of determining competition for theretinal binding pocket, for example, by determining the rate of increasein the 500 nm peak characteristic for rhodopsin. No antibodies forrhodopsin are required for this assay. A useful compound will exhibit arate of rhodopsin formation that is at least about 2 to 5 fold lowerthan that observed in the presence of 11-cis-retinal when said testcompound is not present.

In specific embodiments of the methods of the invention, the misfoldedopsin protein comprises a mutation in its amino acid sequence, forexample, one of the mutations T17M, P347S, R135W or P23H, preferablyP23H.

Preferably, in any of the methods of the invention, the opsin-bindingagent binds to opsin in its retinal binding pocket.

In one aspect, the present invention provides a method of inhibiting theformation or accumulation of a visual cycle product, comprisingcontacting an opsin protein with a compound that reduces hydration ofsaid opsin protein, preferably wherein said compound competes with oneor more water molecules for binding to opsin. In specific embodiments ofsuch methods, the compound binds chemically to the opsin protein, forexample, through hydrogen bonding.

While use of any of the compounds disclosed herein as a means ofreducing hydration in the opsin binding pocket should be considered apreferred embodiment of such method, the reduction of formation of avisual cycle product by reducing the formation of rhodopsin is a generalmethod of the invention for reducing such visual cycle productformation, especially production of lipofuscin and/or A2E, and fortreating an ophthalmic disease by reducing said hydration is a generalaim of the invention and is not necessarily limited in scope only to theuse of chemicals disclosed herein but may include use of other known oryet to be known chemical compounds so long as they function in themethods of the invention and reduce hydration (i.e., binding of water)in the retinal binding pocket of opsin.

It should be noted that the compounds disclosed herein for use in themethods of the invention may not function to reduce hydration in theretinal binding pocket of opsin but may still function in one or more ofthe methods of the invention. For example, a compound of Formula I maybind to an allosteric site on the protein thereby excluding retinal fromthe retinal binding site without necessarily decreasing hydration yetstill reduce formation of a visual cycle product, such as lipofuscinand/or A2E, by virtue of its excluding retinal from the binding pocket,thus non-covalently reducing the activity of the visual cycle.

In embodiments of any of the compositions and methods of the invention,the opsin-binding agent (e.g., a non-retinoid binding agent) isselective for binding to opsin. Such selectivity is not to be taken asrequiring exclusivity that said agent may bind to other proteins as wellas to opsin but its binding to opsin will be at least selective, wherebythe binding constant (or dissociation constant) for binding to opsinwill be lower than the average value for binding to other proteins thatalso bind retinoids, such as retinal analogs. Preferably, opsin bindingagents are non-retinoid opsin-binding agents that bind non-covalently toopsin. Preferably, the opsin binding agent binds at or near the opsinretinal binding pocket, where the native ligand, 11-cis-retinal,normally binds. Without wishing to be bound by theory, in one embodimentthe binding pocket accommodates retinal or an agent of the invention,but not both. Accordingly, when an agent of the invention is bound at ornear the retinal binding pocket, other retinoids, such as11-cis-retinal, are unable to bind to opsin. Binding of an agent of theinvention inside the retinal binding pocket of a misfolded opsinmolecule serves to direct formation of the native or wild-typeconformation of the opsin molecule or to stabilize a correctly foldedopsin protein, thereby facilitating insertion of the nowcorrectly-folded opsin into the membrane of a rod cell. Again, withoutwishing to be bound by theory, said insertion may help to maintain thewild-type conformation of opsin and the opsin-binding agent is free todiffuse out of the binding pocket, whereupon the pocket is available forbinding to retinal to form light-sensitive rhodopsin.

Other methods of the invention provide a means to restore photoreceptorfunction in a mammalian eye containing a misfolded opsin protein thatcauses reduced photoreceptor function, comprising contacting saidmisfolded opsin protein with an opsin-binding agent (e.g., anon-retinoid) that reversibly binds (e.g., that binds non-covalently) ator near the retinal binding pocket. In other embodiments, binding of theopsin-binding agent to the misfolded opsin protein competes with11-cis-retinal for binding in said binding pocket. Desirably, binding ofthe opsin-binding agent restores the native conformation of saidmisfolded opsin protein.

In preferred embodiments, the mammalian eye is a human eye. Inadditional embodiments, said contacting occurs by administering saidopsin-binding agent (e.g., non-retinoid) to a mammal afflicted with anophthalmic condition, such as a condition characterized by reducedphotoreceptor function. In various embodiments, the condition is the wetor dry form of macular degeneration, diabetic RP, a retinal or maculardystrophy, Stargardt's disease, Sorsby's dystrophy, autosomal dominantdrusen, Best's dystrophy, peripherin mutation associate with maculardystrophy, dominant form of Stargart's disease, North Carolina maculardystrophy, light toxicity (e.g., due to retinal surgery), or retinitispigmentosa. The administration may be topical administration or bysystemic administration, the latter including oral administration,intraocular injection or periocular injection. Topical administrationcan include, for example, eye drops containing an effective amount of anagent of the invention in a suitable pharmaceutical carrier.

In another embodiment, the present invention also provides a method ofstabilizing a mutant opsin protein, comprising contacting said mutantopsin protein with a non-retinoid opsin-binding agent that reversiblybinds non-covalently (for example, at or in the retinal binding pocket)to said mutant opsin protein to prevent retinoid binding in said bindingpocket, thereby stabilizing said mutant opsin protein.

The present invention also provides a method of ameliorating loss ofphotoreceptor function in a mammalian eye, comprising administering aneffective amount of an opsin-binding agent, such as a non-retinoid, to amammal afflicted with a mutant opsin protein that has reduced affinityfor 11-cis-retinal, whereby the opsin binding agent reversibly binds(e.g., non-covalently) to the retinal binding pocket of said mutantopsin, thereby ameliorating loss of photoreceptor function in saidmammalian eye. In one embodiment, the contacting occurs by administeringsaid opsin-binding agent to a mammal afflicted with said reducedphotoreceptor function, wherein said administering may be by topicaladministration or by systemic administration, the latter including oral,intraocular injection or periocular injection, and the former includingthe use of eye drops containing an agent of the invention. Such loss ofphotoreceptor function may be a partial loss or a complete loss, andwhere a partial loss it may be to any degree between 1% loss and 99%loss. In addition, such loss may be due to the presence of a mutationthat causes misfolding of the opsin, such as where the mutation is theP23H mutation. In another embodiment, the opsin binding agent isadministered to ameliorate an ophthalmic condition related to themislocalization of an opsin protein. In one embodiment, the inventionprovides for the treatment of a subject having the dry form ofage-related macular degeneration, where at least a portion of the opsinpresent in an ocular photoreceptor cell (e.g., a rod or cone cell) ismislocalized. The mislocalized protein fails to be inserted into themembrane of a photoreceptor cell, where its function is required forvision. Administration of the opsin binding agent to a subject having amislocalized opsin protein rescues, at least in part, opsinlocalization. Accordingly, the invention is useful to prevent or treatan ophthalmic condition related to opsin mislocalization or toameliorate a symptom thereof.

The present invention provides a method for treating and/or preventingan ophthalmic condition or a symptom thereof, including but not limitedto, wet or dry form of macular degeneration, retinitis pigmentosa, aretinal or macular dystrophy, Stargardt's disease, Sorsby's dystrophy,autosomal dominant drusen, Best's dystrophy, peripherin mutationassociate with macular dystrophy, dominant form of Stargart's disease,North Carolina macular dystrophy, light toxicity (e.g., due to retinalsurgery), or retinitis pigmentosa in a subject, such as a human patient,comprising administering to a subject afflicted with, or at risk ofdeveloping, one of the aforementioned conditions or another ophthalmiccondition related to the expression of a misfolded or mislocalized opsinprotein using a therapeutically effective amount of an opsin-bindingagent, e.g., an agent that shows positive activity when tested in anyone or more of the screening assays of the invention.

Such a method may also comprise administering to said subject at leastone additional agent selected from the group consisting of a proteasomalinhibitor, an autophagy inhibitor, a lysosomal inhibitor, an inhibitorof protein transport from the ER to the Golgi, an Hsp90 chaperoneinhibitor, a heat shock response activator, a glycosidase inhibitor, anda histone deacetylase inhibitor, wherein the opsin-binding compound andthe additional compound are administered simultaneously or withinfourteen days of each other in amounts sufficient to treat the subject.

Here again the patient may comprise a mutation that affects proteinfolding where said mutation(s) causes misfolding, e.g., in an opsinprotein, and may be any of the mutations recited elsewhere herein, suchas a P23H mutation. In other embodiments, the patient has an ophthalmiccondition that is related to the mislocalization of an opsin protein.The mislocalized opsin fails to insert into the membrane of aphotoreceptor cell (e.g., a rod or cone cell). In general, this failurein localization would effect only a portion of the opsin present in anocular cell of a patient.

In particular examples of the methods of the invention, theopsin-binding compound and the additional compound are administeredwithin ten days of each other, more preferably within five days of eachother, even more preferably within twenty-four hours of each other andmost preferably are administered simultaneously. In one example, theopsin-binding compound and the additional compound are administereddirectly to the eye. Such administration may be intra-ocular. In otherexamples, the opsin-binding compound and the additional compound areeach incorporated into a composition that provides for their long-termrelease, such as where the composition is part of a microsphere,nanosphere, or nano emulsion. In one example, the composition isadministered via a drug-delivery device that effects long-term release.Such methods also contemplate administering a vitamin A supplement alongwith an agent of the invention.

As described herein, the opsin-binding agents useful in the methods ofthe invention are available for use alone or in combination with one ormore additional compounds to treat or prevent conditions associated withthe wet or dry form of macular degeneration, retinitis pigmentosa, aretinal or macular dystrophy, Stargardt's disease, Sorsby's dystrophy,autosomal dominant drusen, Best's dystrophy, peripherin mutationassociate with macular dystrophy, dominant form of Stargart's disease,North Carolina macular dystrophy, light toxicity (e.g., due to retinalsurgery), retinitis pigmentosa or another ophthalmic condition relatedto the expression of a misfolded or mislocalized opsin protein. In oneembodiment, an opsin-binding compound of the invention (e.g., anon-retinoid or a retinoid that fails to covalently bind to opsin) isadministered to a subject identified as having or at risk of developingsuch a condition. Optionally, the opsin binding agent is administeredtogether with another therapeutic agent. In another embodiment, anon-retinoid opsin-binding compound of the invention is used incombination with a synthetic retinoid (e.g., as disclosed in U.S. PatentPublication No. 2004-0242704), and optionally with another activecompound (e.g., as discussed herein). In still another exemplaryembodiment, an opsin-binding compound is administered in combinationwith the proteasomal inhibitor MG132, the autophagy inhibitor3-methyladenine, a lysosomal inhibitor, such as ammonium chloride, theER-Golgi transport inhibitor brefeldin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and/or the histone deacetylase inhibitor Scriptaid, or anyother agent that can stabilize a mutant P23H opsin protein in abiochemically functional conformation that allows it to associate with11-cis-retinal to form rhodopsin.

In specific embodiments, an opsin-binding compound is a non-polymeric(e.g., a small molecule, such as those disclosed herein for use in themethods of the invention) compound having a molecular weight less thanabout 1000 daltons, less than 800, less than 600, less than 500, lessthan 400, or less than about 300 daltons. In certain embodiments, acompound of the invention increases the amount (e.g., from or in a cell)of a stably-folded and/or complexed mutant protein by at least 10%, 15%,20%, 25%, 50%, 75%, or 100% compared to an untreated control cell orprotein.

Proteasomal Inhibitors

The 26S proteasome is a multicatalytic protease that cleaves ubiquinatedproteins into short peptides. MG-132 is one proteasomal inhibitor thatmay be used. MG-132 is particularly useful for the treatment of lighttoxicity and other ocular diseases related to the accumulation of visualcycle products (e.g., all-trans-retinal, A2E, lipofuscin), proteinaggregation or protein misfolding. Other proteasomal inhibitors usefulin combination with of the invention in the methods of the inventioninclude lactocystin (LC), clasto-lactocystin-beta-lactone, PSI(N-carbobenzoyl-lle-Glu-(OtBu)-Ala-Leu-CHO (SEQ ID NO: 1)), MG-132(N-carbobenzoyl-Leu-Leu-Leu-CHO), MG-115(N-carbobenzoyl-Leu-Leu-Nva-CHO), MG-101 (N-Acetyl-Leu-Leu-norLeu-CHO),ALLM (N-Acetyl-Leu-Leu-Met-CHO), N-carbobenzoyl-Gly-Pro-Phe-leu-CHO (SEQID NO: 2), N-carbobenzoyl-Gly-Pro-Ala-Phe-CHO (SEQ ID NO: 3),N-carbobenzoyl-Leu-Leu-Phe-CHO, and salts or analogs thereof. Otherproteasomal inhibitors and their uses are described in U.S. Pat. No.6,492,333.

Autophagy Inhibitors

Autophagy is an evolutionarily conserved mechanism for the degradationof cellular components in the cytoplasm, and serves as a cell survivalmechanism in starving cells. During autophagy pieces of cytoplasm becomeencapsulated by cellular membranes, forming autophagic vacuoles thateventually fuse with lysosomes to have their contents degraded.Autophagy inhibitors may be used in combination with an opsin-binding oropsin-stabilizing compound of the invention. Autophagy inhibitors usefulin combination with a of the invention in the methods of the inventioninclude, but are not limited to, 3-methyladenine, 3-methyl adenosine,adenosine, okadaic acid, N⁶-mercaptopurine riboside (N⁶-MPR), anaminothiolated adenosine analog, 5-amino-4-imidazole carboxamideriboside (AICAR), bafilomycin A1, and salts or analogs thereof.

Lysosomal Inhibitors

The lysosome is a major site of cellular protein degradation.Degradation of proteins entering the cell by receptor-mediatedendocytosis or by pinocytosis, and of plasma membrane proteins takesplace in lysosomes. Lysosomal inhibitors, such as ammonium chloride,leupeptin, trans-epoxysaccinyl-L-leucylamide-(4-guanidino) butane,L-methionine methyl ester, ammonium chloride, methylamine, chloroquine,and salts or analogs thereof, are useful in combination with anopsin-binding or opsin-stabilizing compound of the invention.

HSP90 Chaperone Inhibitors

Heat shock protein 90 (Hsp90) is responsible for chaperoning proteinsinvolved in cell signaling, proliferation and survival, and is essentialfor the conformational stability and function of a number of proteins.HSP-90 inhibitors are useful in combination with an opsin-binding oropsin-stabilizing compound in the methods of the invention. HSP-90inhibitors include benzoquinone ansamycin antibiotics, such asgeldanamycin and 17-allylamino-17-demethoxygeldanamycin (17-AAG), whichspecifically bind to Hsp90, alter its function, and promote theproteolytic degradation of substrate proteins. Other HSP-90 inhibitorsinclude, but are not limited to, radicicol, novobiocin, and any Hsp90inhibitor that binds to the Hsp90 ATP/ADP pocket.

Heat Shock Response Activators

Celastrol, a quinone methide triterpene, activates the human heat shockresponse. In combination with an opsin-binding or opsin-stabilizingcompound in methods of the invention, celastrol and other heat shockresponse activators are useful for the treatment of PCD. Heat shockresponse activators include, but are not limited to, celastrol,celastrol methyl ester, dihydrocelastrol diacetate celastrol butylester, dihydrocelastrol, and salts or analogs thereof.

Histone Deacetylase Inhibitors

Regulation of gene expression is mediated by several mechanisms,including the post-translational modifications of histones by dynamicacetylation and deacetylation. The enzymes responsible for reversibleacetylation)/deacetylation processes are histone acetyltransferases(HATS) and histone deacetylases (HDACs), respectively. Histonedeacetylase inhibitors include Scriptaid, APHA Compound 8, Apicidin,sodium butyrate, (−)-Depudecin, Sirtinol, trichostatin A, and salts oranalogs thereof. Such inhibitors may be used in combination withcompounds of the invention in the methods disclosed herein.

Glycosidase Inhibitors

Glycosidase inhibitors are one class of compounds that are useful in themethods of the invention, when administered in combination with anopsin-binding or opsin-stabilizing compound of the invention.Castanospermine, a polyhydroxy alkaloid isolated from plant sources,inhibits enzymatic glycoside hydrolysis. Castanospermine and itsderivatives are particularly useful for the treatment of light toxicityor of an ocular Protein Conformation Disorder, such as RP. Also usefulin the methods of the invention are other glycosidase inhibitors,including australine hydrochloride,6-Acetamido-6-deoxy-castanosperrnine, which is a powerful inhibitor ofhexosaminidases, Deoxyfuconojirimycin hydrochloride (DF J7),Deoxynojirimycin (DNJ), which inhibits glucosidase I and II,Deoxygalactonojirimycin hydrochloride (DGJ), winch inhibitsα-D-galactosidase, Deoxymannojirimycin hydrochloride (DMI),2R,5R-Bis(hydroxymethyl)-3R,4R-dihydroxypyrrolidine (DMDP), also knownas 2,5-dideoxy-2,5-imino-D-mannitol, 1,4-Dideoxy-1,4-imino-D-mannitolhydrochloride, (3R,4R,5R,6R)-3,4,5,6-Tetrahydroxyazepane Hydrochloride,which inhibits b-N-acetylglucosaminidase, 1,5-Dideoxy-1,5-imino-xylitol,which inhibits β-glucosidase, and Kifunensine, an inhibitor ofmannosidase 1. Also useful in combination with an opsin-binding oropsin-stabilizing compound are N-butyldeoxynojirimycin (EDNJ), N-nonylDNJ (NDND, N-hexyl DNJ (I5TDNJ), N-methyldeoxynojirimycin (MDNJ), andother glycosidase inhibitors known in the art. Glycosidase inhibitorsare available commercially, for example, from Industrial ResearchLimited (Wellington, New Zealand) and methods of using them aredescribed, for example, in U.S. Pat. Nos. 4,894,388, 5,043,273,5,103,008, 5,844,102, and 6,831,176; and in U.S. Patent Publication Nos.20020006909.

Pharmaceutical Compositions

The present invention features pharmaceutical preparations comprisingcompounds together with pharmaceutically acceptable carriers, where thecompounds provide for the inhibition of visual cycle products, such asall-trans-retinal or other products formed from 11-cis-retinal. Suchpreparations have both therapeutic and prophylactic applications. In oneembodiment, a pharmaceutical composition includes an opsin-binding orstabilizing compound (e.g., a compound identified using the methods ofExample 1) or a pharmaceutically acceptable salt thereof; optionally incombination with at least one additional compound that is a proteasomalinhibitor, an autophagy inhibitor, a lysosomal inhibitor, an inhibitorof protein transport from the ER to the Golgi, an Hsp9O chaperoneinhibitor, a heat shock response activator, a glycosidase inhibitor, ora histone deacetylase inhibitor. The opsin-binding or opsin-stabilizingcompound is preferably not a natural or synthetic retinoid. Theopsin-binding or opsin-stabilizing compound and the additional compoundare formulated together or separately. Compounds of the invention may beadministered as part of a pharmaceutical composition. The non-oralcompositions should be sterile and contain a therapeutically effectiveamount of the opsin-binding or opsin-stabilizing compound in a unit ofweight or volume suitable for administration to a subject. Thecompositions and combinations of the invention can be part of apharmaceutical pack, where each of the compounds is present inindividual dosage amounts.

The phrase “pharmaceutically acceptable” refers to those compounds ofthe present invention, compositions containing such compounds, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

Non-oral pharmaceutical compositions of the invention to be used forprophylactic or therapeutic administration should be sterile. Sterilityis readily accomplished by filtration through sterile filtrationmembranes (e.g., 0.2 μm membranes), by gamma irradiation, or any othersuitable means known to those skilled in the art. Therapeuticopsin-binding or opsin-stabilizing compound compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle. These compositions ordinarily will bestored in unit or multi-dose containers, for example, sealed ampoules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution. The compounds may be combined, optionally, with apharmaceutically acceptable excipient.

The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction that wouldsubstantially impair the desired pharmaceutical efficacy.

Compounds of the present invention can be contained in apharmaceutically acceptable excipient. The excipient preferably containsminor amounts of additives such as substances that enhance isotonicityand chemical stability. Such materials are non-toxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, succinate, acetate, lactate, tartrate, and otherorganic acids or their salts; tris-hydroxymethylaminomethane (TRIS),bicarbonate, carbonate, and other organic bases and their salts;antioxidants, such as ascorbic acid; low molecular weight (for example,less than about ten residues) polypeptides, e.g., polyarginine,polylysine, polyglutamate and polyaspartate; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers, such aspolyvinylpyrrolidone (PVP), polypropylene glycols (PPGs), andpolyethylene glycols (PEGs); amino acids, such as glycine, glutamicacid, aspartic acid, histidine, lysine, or arginine; monosaccharides,disaccharides, and other carbohydrates including cellulose or itsderivatives, glucose, mannose, sucrose, dextrins or sulfatedcarbohydrate derivatives, such as heparin, chondroitin sulfate ordextran sulfate; polyvalent metal ions, such as divalent metal ionsincluding calcium ions, magnesium ions and manganese ions; chelatingagents, such as ethylenediamine tetraacetic acid (EDTA); sugar alcohols,such as mannitol or sorbitol; counterions, such as sodium or ammonium;and/or nonionic surfactants, such as polysorbates or poloxamers. Otheradditives may be included, such as stabilizers, anti-microbials, inertgases, fluid and nutrient replenishers (i.e., Ringer's dextrose),electrolyte replenishers, which can be present in conventional amounts.

The compositions, as described above, can be administered in effectiveamounts. The effective amount will depend upon the mode oradministration, the particular condition being treated and the desiredoutcome. It may also depend upon the stage of the condition, the age andphysical condition of the subject, the nature of concurrent therapy, ifany, and like factors well known to the medical practitioner. Fortherapeutic applications, it is that amount sufficient to achieve amedically desirable result.

With respect to a subject suffering from, or at risk of developing,light toxicity, such as that due to ocular surgery, an effective amountis an amount sufficient to reduce the rate or extent of formation andaccumulation of visual cycle products, such as all-trans-retinal, orlipofuscin, or A2E as well as preventing photocell apoptosis as a resultof excessive rhodopsin activation. Here, the compounds of the presentinvention would be from about 0.01 mg/kg per day to about 1000 mg/kg perday. It is expected that doses ranging from about 50 to about 2000 mg/kgwill be suitable. Lower doses will result from certain forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of a composition of the present invention.

A variety of administration routes are available. The methods of theinvention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. In one preferred embodiment, acomposition of the invention is administered intraocularly. Other modesof administration include oral, rectal, topical, intraocular, buccal,intravaginal, intracisternal, intracerebroventricular, intratracheal,nasal, transdermal, within/on implants, or parenteral routes.Compositions comprising a composition of the invention can be added to aphysiological fluid, such as to the intravitreal humor. For CNSadministration, a variety of techniques are available for promotingtransfer of the therapeutic across the blood brain barrier includingdisruption by surgery or injection, drugs which transiently openadhesion contact between the CNS vasculature endothelial cells, andcompounds that facilitate translocation through such cells. Oraladministration can be preferred for prophylactic treatment because ofthe convenience to the patient as well as the dosing schedule.

Pharmaceutical compositions of the invention can optionally furthercontain one or more additional proteins as desired, including plasmaproteins, proteases, and other biological material, so long as it doesnot cause adverse effects upon administration to a subject. Suitableproteins or biological material may be obtained from human or mammalianplasma by any of the purification methods known and available to thoseskilled in the art; from supernatants, extracts, or lysates ofrecombinant tissue culture, viruses, yeast, bacteria, or the like thatcontain a gene that expresses a human or mammalian plasma protein whichhas been introduced according to standard recombinant DNA techniques; orfrom the fluids (e.g., blood, milk, lymph, urine or the like) ortransgenic animals that contain a gene that expresses a human plasmaprotein which has been introduced according to standard transgenictechniques.

Pharmaceutical compositions of the invention can comprise one or more pHbuffering compounds to maintain the pH of the formulation at apredetermined level that reflects physiological pH, such as in the rangeof about 5.0 to about 8.0 (e.g., 6.0, 6.5, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.8). The pH buffering compound used in the aqueousliquid formulation can be an amino acid or mixture of amino acids, suchas histidine or a mixture of amino acids such as histidine and glycine.Alternatively, the pH buffering compound is preferably an agent whichmaintains the pH of the formulation at a predetermined level, such as inthe range of about 5.0 to about 8.0, and which does not chelate calciumions. Illustrative examples of such pH buffering compounds include, butare not limited to, imidazole and acetate ions. The pH bufferingcompound may be present in any amount suitable to maintain the pH of theformulation at a predetermined level.

Pharmaceutical compositions of the invention can also contain one ormore osmotic modulating agents, i.e., a compound that modulates theosmotic properties (e.g., tonicity, osmolality and/or osmotic pressure)of the formulation to a level that is acceptable to the blood stream andblood cells of recipient individuals. The osmotic modulating agent canbe an agent that does not chelate calcium ions. The osmotic modulatingagent can be any compound known or available to those skilled in the artthat modulates the osmotic properties of the formulation. One skilled inthe art may empirically determine the suitability of a given osmoticmodulating agent for use in the inventive formulation. Illustrativeexamples of suitable types of osmotic modulating agents include, but arenot limited to: salts, such as sodium chloride and sodium acetate;sugars, such as sucrose, dextrose, and mannitol; amino acids, such asglycine; and mixtures of one or more of these agents and/or types ofagents. The osmotic modulating agent(s) maybe present in anyconcentration sufficient to modulate the osmotic properties of theformulation.

Compositions comprising an opsin-binding or opsin-stabilizing compoundof the present invention can contain multivalent metal ions, such ascalcium ions, magnesium ions and/or manganese ions. Any multivalentmetal ion that helps stabilize the composition and that will notadversely affect recipient individuals may be used. The skilled artisan,based on these two criteria, can determine suitable metal ionsempirically and suitable sources of such metal ions are known, andinclude inorganic and organic salts.

Pharmaceutical compositions of the invention can also be a non-aqueousliquid formulation. Any suitable non-aqueous liquid may be employed,provided that it provides stability to the active agents (a) containedtherein. Preferably, the non-aqueous liquid is a hydrophilic liquid.Illustrative examples of suitable non-aqueous liquids include: glycerol;dimethyl sulfoxide (DMSO); polydimethylsiloxane (PMS); ethylene glycols,such as ethylene glycol, diethylene glycol, triethylene glycol,polyethylene glycol (“PEG”) 200, PEG 300, and PEG 400; and propyleneglycols, such as dipropylene glycol, tripropylene glycol, polypropyleneglycol (“PPG”) 425, PPG 725, PPG 1000, PEG 2000, PEG 3000 and PEG 4000.

Pharmaceutical compositions of the invention can also be a mixedaqueous/non-aqueous liquid formulation. Any suitable non-aqueous liquidformulation, such as those described above, can be employed along withany aqueous liquid formulation, such as those described above, providedthat the mixed aqueous/non-aqueous liquid formulation provides stabilityto the compound contained therein. Preferably, the non-aqueous liquid insuch a formulation is a hydrophilic liquid. Illustrative examples ofsuitable non-aqueous liquids include: glycerol; DMSO; EMS; ethyleneglycols, such as PEG 200, PEG 300, and PEG 400; and propylene glycols,such as PPG 425, PPG 725, PEG 1000, PEG 2000, PEG 3000 and PEG 4000.Suitable stable formulations can permit storage of the active agents ina frozen or an unfrozen liquid state. Stable liquid formulations can bestored at a temperature of at least −70° C., but can also be stored athigher temperatures of at least 0° C., or between about 0° C. and about42° C., depending on the properties of the composition. It is generallyknown to the skilled artisan that proteins and polypeptides aresensitive to changes in pH, temperature, and a multiplicity of otherfactors that may affect therapeutic efficacy.

In certain embodiments a desirable route of administration can be bypulmonary aerosol. Techniques for preparing aerosol delivery systemscontaining polypeptides are well known to those of skill in the art.Generally, such systems should utilize components that will notsignificantly impair the biological properties of the antibodies, suchas the paratope binding capacity (see, for example, Sciarra and Cutie,“Aerosols,” in Remington's Pharmaceutical Sciences 18th edition, 1990,pp 1694-1712; incorporated by reference). Those of skill in the art canreadily modify the various parameters and conditions for producingpolypeptide aerosols without resorting to undue experimentation.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of compositions of the invention, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as polylactides (U.S. Pat. No.3,773,919; European Patent No. 58,481), poly(lactide-glycolide),copolyoxalates polycaprolactones, polyesteramides, polyorthoesters,poiyhydroxybutyric acids, such as poly-D-(−)-3-hydroxybutyric acid(European Patent No. 133,988), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, K R. et at, Biopolymers 22: 547-556),poly (2-hydroxyethyl methacrylate) or ethylene vinyl acetate (Langer, etal., J. Biomed. Mater. Res. 15:267-277; Langer, B. Chem. Tech.12:98-105), and polyanhydrides.

Other examples of sustained-release compositions include semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Delivery systems also include non-polymer systems thatare: lipids including sterols such as cholesterol, cholesterol estersand fatty acids or neutral fats such as mono-, di- and tri-glycerides;hydrogel release systems such as biologically-derived bioresorbablehydrogel (i.e., chitin hydrogels or chitosan hydrogels); sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially filled implants; and thelike. Specific examples include, but are not limited to: (a) aerosionalsystems in which the agent is contained in a form within a matrix suchas those described in 13.5. U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034 and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253, and 3,854,480.

Another type of delivery system that can be used with the methods andcompositions of the invention is a colloidal dispersion system.Colloidal dispersion systems include lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.Liposomes are artificial membrane vessels, which are useful as adelivery vector in vivo or in vitro. Large unilamellar vessels (LUV),which range in size from 0.2-4.0 μm, can encapsulate largemacromolecules within the aqueous interior and be delivered to cells ina biologically active form (Fraley, R., and Papahadjopoulos, D TrendsBiochem. Sci. 6: 77-80).

Liposomes can be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Liposomes are commercially available from GibcoBRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed ofcationic lipids such as N-[1-(2,3dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Methods for makingliposomes are well known in the art and have been described in manypublications, for example, in DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. (USA) 82:3688-3692 (1985); K. Hwang et al., Proc. Natl. Acad.Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Liposomes also have beenreviewed by Gregoriadis, G., Trends Biotechnol., 3: 235-241.

Another type of vehicle is a biocompatible microparticle or implant thatis suitable for implantation into the mammalian recipient. Exemplarybioerodible implants that are useful in accordance with this method aredescribed in PCT International application no. PCTIUS/03307 (PublicationNo-WO 95/24929, entitled “Polymeric Gene Delivery System”). PCT/US/0307describes biocompatible, preferably biodegradable polymeric matrices forcontaining an exogenous gene under the control of an appropriatepromoter. The polymeric matrices can be used to achieve sustainedrelease of the exogenous gene or gene product in the subject.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein an agent is dispersed throughout a solidpolymeric matrix) or a microcapsule (wherein an agent is stored in thecore of a polymeric shell). Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Other forms of the polymeric matrix for containing an agent includefilms, coatings, gels, implants, and stents. The size and composition ofthe polymeric matrix device is selected to result in favorable releasekinetics in the tissue into which the matrix is introduced. The size ofthe polymeric matrix further is selected according to the method ofdelivery that is to be used. Preferably, when an aerosol route is usedthe polymeric matrix and composition are encompassed in a surfactantvehicle. The polymeric matrix composition can be selected to have bothfavorable degradation rates and also to be formed of a material, whichis a bioadhesive, to further increase the effectiveness of transfer. Thematrix composition also can be selected not to degrade, but rather torelease by diffusion over an extended period of time. The deliverysystem can also be a biocompatible microsphere that is suitable forlocal, site-specific delivery. Such microspheres are disclosed inChickering, D. B., et al., Biotechnot. Bioeng, 52: 96-101; Mathiowitz,B., et at., Nature 386: 410-414.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the compositions of the invention to the subject. Suchpolymers may be natural or synthetic polymers. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

Exemplary synthetic polymers which can be used to form the biodegradabledelivery system include: polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluoses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetate phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulfate sodium salt,poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate),poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene, poly(ethylene glycol), poly(ethylene oxide),poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate),poly(vinyl chloride), polystyrene, poly(vinyl pyrrolidone), and polymersof lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone),and natural polymers such as alginate and other polysaccharidesincluding dextran and cellulose, collagen, chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), albumin and other hydrophilicproteins, zein and other prolamines and hydrophobic proteins, copolymersand mixtures thereof. In general, these materials degrade either byenzymatic hydrolysis or exposure to water in vivo, by surface or bulkerosion.

Methods of Ocular Delivery

The compositions of the invention are particularly suitable for treatingocular diseases or conditions, such as light toxicity, in particularlight toxicity related to an ocular surgical procedure.

In one approach, the compositions of the invention are administeredthrough an ocular device suitable for direct implantation into thevitreous of the eye. The compositions of the invention may be providedin sustained release compositions, such as those described in, forexample, U.S. Pat. Nos. 5,672,659 and 5,595,760. Such devices are foundto provide sustained controlled release of various compositions to treatthe eye without risk of detrimental local and systemic side effects. Anobject of the present ocular method of delivery is to maximize theamount of drug contained in an intraocular device or implant whileminimizing its size in order to prolong the duration of the implant.See, e.g., U.S. Pat. Nos. 5,378,475; 6,375,972, and 6,756,058 and U.S.Publications 20050096290 and 200501269448. Such implants may bebiodegradable and/or biocompatible implants, or may be non-biodegradableimplants.

Biodegradable ocular implants are described, for example, in U.S. PatentPublication No. 20050048099. The implants may be permeable orimpermeable to the active agent, and may be inserted into a chamber ofthe eye, such as the anterior or posterior chambers or may be implantedin the sclera, transchoroidal space, or an avascularized region exteriorto the vitreous. Alternatively, a contact lens that acts as a depot forcompositions of the invention may also be used for drug delivery.

In a preferred embodiment, the implant may be positioned over anavascular region, such as on the sclera, so as to allow for transcleraldiffusion of the drug to the desired site of treatment, e.g. theintraocular space and macula of the eye. Furthermore, the site oftranscleral diffusion is preferably in proximity to the macula. Examplesof implants for delivery of a composition of the invention include, butare not limited to, the devices described in U.S. Pat. Nos. 3,416,530;3,828,777; 4,014,335; 4,300,557; 4,327,725; 4,853,224; 4,946,450;4,997,652; 5,147,647; 164,188; 5,178,635; 5,300,114; 5,322,691;5,403,901; 5,443,505; 5,466,466; 5,476,511; 5,516,522; 5,632,984;5,679,666; 5,710,165; 5,725,493; 5,743,274; 5,766,242; 5,766,619;5,770,592; 5,773,019; 5,824,072; 5,824,073; 5,830,173; 5,836,935;5,869,079, 5,902,598; 5,904,144; 5,916,584; 6,001,386; 6,074,661;6,110,485; 6,126,687; 6,146.366; 6,251,090; and 6,299,895, and in WO01/30323 and WO 01/28474, all of which are incorporated herein byreference.

Examples include, but are not limited to the following: a sustainedrelease drug delivery system comprising an inner reservoir comprising aneffective amount of an agent effective in obtaining a desired local orsystemic physiological or pharmacological effect, an inner tubeimpermeable to the passage of the agent, the inner tube having first andsecond ends and covering at least a portion of the inner reservoir, theinner tube sized and formed of a material so that the inner tube iscapable of supporting its own weight, an impermeable member positionedat the inner tube first end, the impermeable member preventing passageof the agent out of the reservoir through the inner tube first end, anda permeable member positioned at the inner tube second end, thepermeable member allowing diffusion of the agent out of the reservoirthrough the inner tube second end; a method for administering a compoundof the invention to a segment of an eye, the method comprising the stepof implanting a sustained release device to deliver the compound of theinvention to the vitreous of the eye or an implantable, sustainedrelease device for administering a compound of the invention to asegment of an eye; a sustained release drug delivery device comprising:a) a drug core comprising a therapeutically effective amount of at leastone first agent effective in obtaining a diagnostic effect or effectivein obtaining a desired local or systemic physiological orpharmacological effect; b) at least one unitary cup essentiallyimpermeable to the passage of the agent that surrounds and defines aninternal compartment to accept the drug core, the unitary cup comprisingan open top end with at least one recessed groove around at least someportion of the open top end of the unitary cup; c) a permeable plugwhich is permeable to the passage of the agent, the permeable plug ispositioned at the open top end of the unitary cup wherein the grooveinteracts with the permeable plug holding it in position and closing theopen top end, the permeable plug allowing passage of the agent out ofthe drug core, though the permeable plug, and out the open top end ofthe unitary cup; and d) at least one second agent effective in obtaininga diagnostic effect or effective in obtaining a desired local orsystemic physiological or pharmacological effect; or a sustained releasedrug delivery device comprising: an inner core comprising an effectiveamount of an agent having a desired solubility and a polymer coatinglayer, the polymer layer being permeable to the agent, wherein thepolymer coating layer completely covers the inner core.

Other approaches for ocular delivery include the use of liposomes totarget a compound of the present invention to the eye, and preferably toretinal pigment epithelial cells and/or Bruch's membrane. For example,the compound maybe complexed with liposomes in the manner describedabove, and this compound/liposome complex injected into patients with anophthalmic condition, such as light toxicity, using intravenousinjection to direct the compound to the desired ocular tissue or cell.Directly injecting the liposome complex into the proximity of theretinal pigment epithelial cells or Bruch's membrane can also providefor targeting of the complex with some forms of ocular PCD. In aspecific embodiment, the compound is administered via intra-ocularsustained delivery (such as VITRASERT or ENVISION. In a specificembodiment, the compound is delivered by posterior subtenons injection.In another specific embodiment, microemulsion particles containing thecompositions of the invention are delivered to ocular tissue to take uplipid from Bruchs membrane, retinal pigment epithelial cells, or both.

Nanoparticles are a colloidal carrier system that has been shown toimprove the efficacy of the encapsulated drug by prolonging the serumhalf-life. Polyalkylcyanoacrylates (PACAs) nanoparticles are a polymercolloidal drug delivery system that is in clinical development, asdescribed by Stella et al, J. Pharm. Sci., 2000. 89: p. 1452-1464;Brigger et al., Tnt. J. Pharm., 2001. 214: p. 37-42; Calvo et al.,Pharm. Res., 2001. 18: p. 1157-1166; and Li et al., Biol. Pharm. Bull.,2001. 24: p. 662-665. Biodegradable poly (hydroxyl acids), such as thecopolymers of poly (lactic acid) (PLA) and poly (lactic-co-glycolide)(PLGA) are being extensively used in biomedical applications and havereceived FDA approval for certain clinical applications. In addition,PEG-PLGA nanoparticles have many desirable carrier features including(i) that the agent to be encapsulated comprises a reasonably high weightfraction (loading) of the total carrier system; (ii) that the amount ofagent used in the first step of the encapsulation process isincorporated into the final carrier (entrapment efficiency) at areasonably high level; (iii) that the carrier have the ability to befreeze-dried and reconstituted in solution without aggregation; (iv)that the carrier be biodegradable; (v) that the carrier system be ofsmall size; and (vi) that the carrier enhance the particles persistence.

Nanoparticles are synthesized using virtually any biodegradable shellknown in the art. In one embodiment, a polymer, such as poly(lactic-acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) is used.Such polymers are biocompatible and biodegradable, and are subject tomodifications that desirably increase the photochemical efficacy andcirculation lifetime of the nanoparticle. In one embodiment, the polymeris modified with a terminal carboxylic acid group (COOH) that increasesthe negative charge of the particle and thus limits the interaction withnegatively charge nucleic acid aptamers. Nanoparticles are also modifiedwith polyethylene glycol (PEG), which also increases the half-life andstability of the particles in circulation. Alternatively, the COOH groupis converted to an N-hydroxysuccinimide (NHS) ester for covalentconjugation to amine-modified aptamers.

Biocompatible polymers useful in the composition and methods of theinvention include, but are not limited to, polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, poly(vinyl pyrrolidone), polyglycolides,polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylic and methacrylic esters, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, cellulosesulfate sodium salt poly-methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate\ poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate),polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide),poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate,polyvinyl chloride polystyrene, poly(vinyl pyrrolidone), polyhyaluronicacids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid,alginate, chitosan, poly(methyl methacrylates), poly(ethylmethacrylates), poly(butyl methacrylate), poly(isobutyl methacrylate),poly(hexyl methacrylate) poly(isodecyl methaerylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylatee), poly(isobutyl acrylate), poly(octadecylacrylate) and combinations of any of these, In one embodiment, thenanoparticles of the invention include PEG-PLGA polymers.

Compositions of the invention may also be delivered topically. Fortopical delivery, the compositions are provided in any pharmaceuticallyacceptable excipient that is approved for ocular delivery. Preferably,the composition is delivered in drop form to the surface of the eye. Forsome application, the delivery of the composition relies on thediffusion of the compounds through the cornea to the interior of theeye.

Those of skill in the art will recognize that treatment regimens forusing the compounds of the present invention to treat light toxicity orother ophthalmic conditions (e.g., RP) can be straightforwardlydetermined. This is not a question of experimentation, but rather one ofoptimization, which is routinely conducted in the medical arts. In vivostudies in nude mice often provide a starting point from which to beginto optimize the dosage and delivery regimes. The frequency of injectionwill initially be once a week, as has been done in some mice studies.However, this frequency might be optimally adjusted from one day toevery two weeks to monthly, depending upon the results obtained frontthe initial clinical trials and the needs of a particular patient.

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice, as a skilled artisan recognizes itis routine in the art to modify the dosage for humans compared to animalmodels. For certain embodiments it is envisioned that the dosage mayvary from between about 1 mg compound/Kg body weight to about 5000 mgcompound/Kg body weight; or from about 5 mg/Kg body weight to about 4000mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kgbody weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg bodyweight; or from about 100 mg/Kg body weight to about 1000 mg/Kg bodyweight; or from about 150 mg/Kg body weight to about 500 mg/Kg bodyweight. In other embodiments this dose maybe about 1, 5, 10, 25, 50,75,100, 150, 10 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, 5000 mg/Kg body weight. in other embodiments, it is envisaged thatlower does may be used, such doses may be in the range of about 5 mgcompound/Kg body to about 20 mg compound/Kg body. In other embodimentsthe doses may be about 8, 10, 12, 14, 16 15 or 18 mg/Kg body weight. Ofcourse, this dosage amount may be adjusted upward or downward, as isroutinely done in such treatment protocols, depending on the results ofthe initial clinical trials and the needs of a particular patient.

Screening Assays

Useful compounds of the invention are compounds of the formula (I) thatreversibly bind to a native or mutated opsin protein, such as in or nearthe 11-cis-retinal binding pocket. The non bleachable or slowlybleachable pigment rhodopsins formed from these small molecule opsinbindings will prevent light toxicity related to, for example, theaccumulation of visual cycle products as well as apoptotic photocelldeath resulting from excessive rhodopsin stimulation. Such binding willcommonly inhibit, if not prevent, binding of retinoids, especially11-cis-retinal, to the binding pocket and thereby reduce formation ofvisual cycle products, such as all-trans-retinal. Any number of methodsare available for carrying out screening assays to identify suchcompounds. In one approach, an opsin protein is contacted with acandidate compound or test compound that is a non-retinoid in thepresence of 11-cis-retinal or retinoid analog and the rate or yield offormation of chromophore is determined. If desired, the binding of thenon-retinoid to opsin is characterized. Preferably, the non-retinoidbinding to opsin is non-covalent and reversible. Thus, inhibition ofrhodopsin formation by a non-retinoid indicates identification of asuccessful test compound. An increase in the amount of rhodopsin isassayed, for example, by measuring the protein's absorption at acharacteristic wavelength (e.g., 498 nm for rhodopsin) or by measuringan increase in the biological activity of the protein using any standardmethod (e.g., enzymatic activity association with a ligand). Usefulcompounds inhibit binding of 11-cis-retinal (and formation of rhodopsin)by at least about 10%, 15%, or 20%, or preferably by 25%, 50%, or 75%,or most preferably by up to 90% or even 100%.

Alternatively, the efficacy of compounds useful in the methods of theinvention may be determined by exposure of a mammalian eye to a highintensity light source prior to, during, or following administration ofa test compound, followed by determination of the amount of visual cycleproducts (e.g., all-trans retinal, A2E, or lipofuscin) formed as aresult of exposure to the high intensity light source, wherein acompound of the invention will have reduced the amount of visual cycleproducts related to the exposure.

In sum, preferred test compounds identified by the screening methods ofthe invention are non-retinoids, are selective for opsin and bind in areversible, non-covalent manner to opsin protein. In addition, theiradministration to transgenic animals otherwise producing increasedlipofuscin results in a reduced rate of production or a reducedaccumulation of lipofuscin in the eye of said animal. Compoundsidentified according to the methods of the invention are useful for thetreatment of light toxicity or other ophthalmic condition in a subject,such as a human patient.

Compositions of the invention useful for the prevention of lighttoxicity, as well as AMD and retinitis pigmentosa, can optionally becombined with additional therapies as heretofore described.

EXAMPLES

The following non-limiting examples further describe and enable one ofordinary skill in the art to make use of the invention.

Example 16-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamideExample 1a isoquinolin-6-yl(2,6,6-trimethylcyclohex-1-enyl)methanol

A solution of 6-bromoisoquinoline (274 mg, 1.3 mmol) in drytetrahydrofuran (2 mL) was added dropwise to a solution of n-butyllithium (1.6 M in hexane, 0.8 mL, 1.3 mmol) at −78° C. and stirred atthis temperature for 30 minutes. Then2,6,6-trimethylcyclohex-1-enecarbaldehyde (100 mg, 0.66 mmol) in drytetrahydrofuran (1.5 mL) was added and stirring at −78° C. was continuedfor 1 hour after which the reaction was allowed to warm slowly to roomtemperature. The mixture was quenched with saturated aqueous ammoniumchloride and the organics extracted extracted with ethyl acetate. Thecombined organic phase was washed with brine, dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to afford the titlecompound (131 mg, Yield: 71%). R_(f) 0.5 (2:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 9.22 (s, 1 H), 8.50 (d, J=5.6 Hz, 1H), 7.92 (d, J=8.4 Hz, 2 H), 7.65 (d, J=6.4 Hz, 2 H), 5.55 (s, 1 H),2.00 (t, J=6.0 Hz, 2 H), 1.74-1.56 (m, 5 H), 1.33 (s, 3 H), 1.24 (s 3H), 1.17 (s, 3 H) ppm; Mass spectrum (ESI+ve) m/z 282 (M+⁺).

Example 1b isoquinolin-6-yl(2,6,6-trimethylcyclohex-1-enyl)methanone

To a solution of the product of Example la (130 mg, 0.46 mmol) indichloromethane (5 mL) at 0° C. was added Dess-Martin periodinane (529mg, 1.25 mmol) and the reaction was stirred for 30 minutes. The mixturewas then diluted with petroleum ether and filtered. The filtrate wasconcentrated under reduced pressure and the residue purified purified bysilica gel column chromatography to give the title compound (96 mg,Yield: 74%). R_(f) 0.4 (5:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 9.34 (s, 1 H), 8.62 (d, J=5.6 Hz, 1 H), 8.37 (s, 1 H),8.16 (dd, J₁=8.4 Hz, J₂=1.6 Hz, 1 H), 8.06 (d, J=8.8 Hz, 1 H), 7.79 (d,J=6.0 Hz, 1 H), 2.15 (t, J=6.4 Hz, 2 H), 1.84-1.79 (m, 2 H), 1.63-1.60(m, 2 H), 1.47 (s, 3 H), 1.07 (s, 6 H) ppm.

Example 1c(1,2,3,4-tetrahydroisoquinolin-6-yl)(2,6,6-trimethylcyclohex-1-enyl)methanone

To the product of Example 1 b (87 mg, 0.31 mmol) in acetic acid (2 mL)was added platinum dioxide (14 mg) and the reaction was stirred under anatmosphere of hydrogen for 3 hours at room temperature. The mixture wasfiltered and the filtrate concentrated under reduced pressure. Theresidue was dissolved in ethyl acetate and adjusted to pH=8 withsaturated aqueous sodium bicarbonate and the aqueous layer was extractedwith ethyl acetate. The combined organic phase was washed with brine,dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (76.5 mg, Yield: 86%). R_(f) 0.5 (10:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.70 (s, 1 H), 7.66(d, J=8.0 Hz, 1 H), 7.08 (d, J=7.6 Hz, 1 H), 4.08 (s, 2 H), 3.18 (t,J=6.0 Hz, 2 H), 2.89 (t, J=5.8 Hz, 2 H), 2.08 (t, J=6.4 Hz, 2 H),1.78-1.74 (m, 2 H), 1.56-1.53 (m, 2 H), 1.43 (s, 3 H), 1.03 (s, 6 H)ppm; Mass spectrum (ESI+ve) m/z 284 (M+H⁺).

Example 16-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 1c (30 mg, 0.11 mmol) indichloromethane (2 mL) was added triethylamine (43 mg, 0.42 mmol) andisocyanatotrimethylsilane (38 mg, 0.33 mmol) and the reaction mixturewas stirred at room temperature for 2 days. The mixture was diluted withdichloromethane and then washed with water, saturated aqueous ammoniumchloride, saturated aqueous sodium bicarbonate, brine, dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by prep-TLC to afford the title compound as a whitesolid (23 mg, Yield: 67%). Mp=86.4-87.6° C.; R_(f) 0.6 (10:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.77 (s, 1 H), 7.73(d, J=7.6 Hz, 1 H), 7.21 (d, J=8.0 Hz, 1 H), 4.64 (s, 2 H), 4.52 (bs, 2H), 3.64 (t, J=5.8 Hz, 2 H), 2.97 (t, J=5.8 Hz, 2 H), 2.08 (t, J=6.4 Hz,2 H), 1.78-1.76 (m, 2 H), 1.57-1.54 (m, 2 H), 1.43 (s, 3 H), 1.03 (s, 6H) ppm; Mass spectrum (ESI +ve) m/z 327 (M +H⁺).

Example 26-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)pmethyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 1 (32 mg, 0.098 mmol) intetrahydrofuran (2 mL) at 0° C. was added lithium aluminum hydride (22mg, 0.588 mmol). The reaction was warmed to room temperature and stirredfor 1 hour. The mixture was quenched with saturated aqueous ammoniumchloride and the organics extracted with ethyl acetate. The combinedorganic phase was washed with saturated aqueous sodium bicarbonate,brine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound as a white solid (20 mg,Yield: 62%). Mp=88.3-89.0° C.; R_(f) 0.55 (10:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.26 (s, 1 H), 7.20(d, J=8.0 Hz, 1 H), 7.06 (d, J=8.0 Hz, 1 H), 5.38 (d, J=4.4 Hz, 1 H),4.56 (s, 2 H), 4.50 (bs, 2 H), 3.63 (t, J=6.0 Hz, 2 H), 2.89 (t, J=5.8Hz, 2 H), 1.99 (t, J=6.0 Hz, 2 H), 1.83 (d, J=5.2 Hz, 1 H), 1.68-1.64(m, 2 H), 1.56-1.52 (m, 2 H), 1.40 (s, 3 H), 1.19 (s, 3 H), 1.05 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 329 (M+H^(+).)

Example 36-((S)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide Example 3a (S)-3,7-dimethylocta-1,6-dienyl acetate

A 50 mL round-bottom-flask equipped with a condenser was charged withacetic anhydride (6.1 mL, 64.8 mmol), potassium acetate (0.51 g, 5.18mmol) and triethylamine (4.5 mL, 32.4 mmol). To the stirred mixture was(S)-3,7-dimethyloct-6-enal (5.0 g, 32.4 mmol) slowly. The reactionmixture was heated to 120° C. for 7.5 hours. After cooling to roomtemperature, the reaction mixture was poured into water (25 mL) and thenextracted with toluene (10 mL). The organic layer was washed withsaturated aqueous sodium carbonate (25 mL×2) and brine (25 mL). Thematerial was transferred to a 50 ml round-bottom-flask and theseparatory funnel was washed with toluene (1 mL). The solution of thetitle compound in toluene (11 ml) (15.7 g) was used directly for thenext step. Mass spectrum (ESI+ve) m/z 187 (M+H⁺).

Example 3b (1R,6S)-2,2,6-trimethylcyclohexanecarbaldehyde

A solution of crude product of Example 3a (6.36 g, 32.4 mmol) in toluene(11 mL) (15.7 g) was added 85% phosphoric acid (12 mL). The mixture washeated to 100° C. for 4 hours. The reaction mixture was cooled to roomtemperature and toluene (12 mL) along with water (24 mL) was added andthe layers were separated. The aqueous layer was extracted with toluene(12 mL×2). The combined organic layers were washed with saturatedaqueous sodium bicarbonate (50 mL×2) and brine (50 mL×2). Concentrationand distillation under reduced pressure (b.p. 65-70° C.) afforded thetitle compound as a 9:1 mixture of epimers as a colorless oil (2.51 g,Yield: 50%). ¹H NMR (400 MHz, CDCl₃) (Major) δ 9.63 (d, J=5.2 Hz, 1H),2.03-1.91 (m, 1H), 1.83-1.75 (m, 1H), 1.64-1.60 (m, 1H), 1.54-1.48 (m,1H), 1.40-1.35 (m, 1H), 1.24-1.14 (m, 1H), 1.02 (s, 3H), 0.97 (s, 3H),0.95-0.84 (m, 2H), 0.81 (d, J=6.4 Hz, 3H) ppm. [α]_(D) ²⁴=+5.20°(c=1.00, dichloromethane).

Example 3c(S)-isoquinolin-6-yl((1R,6S)-2,2,6-trimethylcyclohexyl)methanol

A solution of 6-bromoisoquinoline (2.08 g, 10 mmol) in drytetrahydrofuran (20 mL) at −78° C. was added dropwise to n-butyl lithium(1.6 M in hexane, 6.25 mL, 10 mmol) and stirred at −78° C. for 15minutes. The product of Example 3b (770 mg, 5 mmol) in tetrahydrofuran(5 mL) was added and stirred at −78° C. for 30 minutes and then slowlywarmed to room temperature and stirred for an additional 2 hours. Themixture was quenched with saturated aqueous ammonium chloride and theorganics were extracted with ethyl acetate. The combined organic phasewas washed with brine, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. Purification of residue by silicagel column chromatography afforded a yellow solid. The solid wasrecrystallized from petroleum ether and ethyl acetate to give the titlecompound as a white solid (778 mg, Yield: 55%). R_(f)=0,4 (2:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 9.20 (s, 1 H), 8.49 (d,J=5.6 Hz, 1 H), 7.93-7.90 (m, 2 H), 7.64-7.60 (m, 2 H), 5.28 (d, J=5.6Hz, 1 H), 2.02 (d, J=5.6 Hz, 1 H), 1.91-1.88 (m, 1 H), 1.56-1.47 (m, 4H), 1.37-1.26 (m, 2 H), 1.23 (s, 3 H), 1.12 (s, 3 H), 1.01-0.93 (m, 1H), 0.56 (d, J=6.4 Hz, 3 H) ppm; Mass spectrum (ESI+ve) m/z 284 (M+H⁺).

Example 3d(S)-(1,2,3,4-tetrahydroisoquinolin-6-yl)((1R,6S)-2,2,6-trimethylcyclohexyl)methanol

To the product of Example 3c (150 mg, 0.53 mmol) in acetic acid (10 mL)was added platinum dioxide (68 mg) and stirred under a hydrogenatmosphere (174 psi) for 1 hour at room temperature. The mixture wasfiltered and the filtrate adjusted to pH=10 with saturated aqueoussodium carbonate. The organics were extracted with ethyl acetate and thecombined organic phase was washed with brine, dried over anhydroussodium sulfate and concentrated under reduced pressure. Purification ofthe residue by column chromatography on neutral alumina afforded thetitle compound (115 mg, Yield: 75%). R_(f) 0.5 (10:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.14-7.10 (m, 2 H),6.94 (d, J=8.0 Hz, 1 H), 5.05 (s, 1 H), 3.99 (s, 2 H), 3.14 (t, J=6.0Hz, 2 H), 2.80 (t, J=5.8 Hz, 2 H), 1.85-1.78 (m, 1 H), 1.67-1.60 (m, 2H), 1.51-1.39 (m, 4 H), 1.32-1.24 (m, 2 H), 1.13 (s, 3 H), 1.06 (s, 3H), 1.00-0.89 (m, 1 H), 0.61 (d, J=6.4 Hz, 3 H) ppm.

Example 36-(S)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 3d (114 mg, 0.4 mmol) indichloromethane (5 mL) was added triethylamine (162 mg, 1.6 mmol) andisocyanatotrimethylsilane (138 mg, 1.2 mmol). The reaction mixture wasstirred at room temperature overnight. The mixture was diluted withdichloromethane and washed with water, saturated aqueous ammoniumchloride, saturated aqueous sodium bicarbonate, brine, dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by prep-TLC to afford the title compound as a whitesolid (93 mg, Yield: 71%). Mp=99.1-100.6° C.; R_(f) 0.5 (15:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.23 (d, J=8.0 Hz,1 H), 7.19 (s, 1 H), 7.07 (d, J=8.0 Hz, 1 H), 5.08 (d, J=5.2 Hz, 1 H),4.55 (s, 2 H), 4.53 (s, 2 H), 3.63 (t, J=5.8 Hz, 2 H), 2.89 (t, J=5.8Hz, 2 H), 1.84-1.81 (m, 1 H), 1.76 (d, J=6.0 Hz, 1 H), 1.50-1.40 (m, 4H), 1.32-1.24 (m, 1 H), 1.14 (s, 3 H), 1.06 (s, 3 H) 0.98-0.93 (m, 1 H),0.60 (d, J=6.4 Hz, 3 H) ppm; Mass spectrum (ESI+ve) m/z 331 (M+H⁺);[α]_(D) ²⁵=+7.41° (c=0.54, dichloromethane).

Example 46-((1R,6S)-2,2,6-trimethylcyclohexanecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 3 (80 mg, 0.24 mmol) indichloromethane (2 mL) at 0° C. was added Dess-Martin periodinane (154mg, 0.36 mmol) and the reaction was stirred for 1 hour. The mixture wasquenched with saturated aqueous sodium carbonate and the organics wereextracted with dichloromethane. The combined organic phase was washedwith brine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by preparative thin layerchromatography (prep-TLC) to give the title compound as a white solid(42 mg, Yield: 53%). Mp=89.1-90.0° C.; R_(f) 0.4 (20:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.80 (d, J=8.0 Hz,1 H), 7.77 (s, 1 H), 7.22 (d, J=8.0 Hz, 1 H), 4.63 (s, 2 H), 4.54 (s, 2H), 3.65 (t, J=5.8 Hz, 2 H), 3.01-2.96 m, 3 H), 2.09-2.06 (m, 1 H), 1.79(dd, J₁=13.2 Hz, J₂=3.2 Hz, 1 H), 1.58-1.54 (m, 2 H), 1.45-1.41 (m, 1H), 1.34-1.25 (m, 1 H), 1.04-1.00 (m, 1 H), 0.98 (s, 3 H), 0.77 (s, 3H), 0.74 (d, J=6.4 Hz, 3 H) ppm; Mass spectrum (ESI+ve) m/z 329 (M+H⁺);[α]_(D) ^(26.6)=−5.33° (c=0.15, dichloromethane).

Example 56-(R)-hydroxy((1R6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 4 (32 mg, 0.1 mmol) intetrahydrofuran (2 mL) at 0° C. was added lithium aluminum hydride (23mg, 0.6 mmol) and the reaction was stirred for 1 hour. The mixture wasquenched with saturated aqueous ammonium chloride and the organics wereextracted with ethyl acetate. The combined organic phase was washed withsaturated aqueous sodium bicarbonate, brine, dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waspurified by prep-TLC to afford the title compound and its epimer (7.7:1)as a white solid (17 mg, Yield: 48%). Mp=77.6-78.5° C.; R_(f) 0.5 (15:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) (Major isomer) δ7.24-7.22 (m, 2 H), 7.07 (d, J=8.0 Hz, 1 H), 5.23 (s, 1 H), 4.56 (s, 2H), 4.51 (bs, 2 H), 3.63 (t, J=5.8 Hz, 2 H), 2.89 (t, J=5.8 Hz, 2 H),1.90-1.84 (m, 1 H), 1.76-1.67 (m, 2 H), 1.47-1.37 (m, 3 H), 1.21-1.10(m, 2 H), 1.06 (d, J=6.0 Hz, 3 H), 1.03 (s, 3 H), 0.41 (s, 3 H) ppm;Mass spectrum (ESI+ve) m/z 331 (M+H⁺); [α]_(D) ^(26.6)=+21.0° (c=0.21,dichloromethane).

Example 67-(2,6,6-trimethylcyclohex-1-enecarbanyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide Example 6aisoquinolin-7-yl(2,6,6-trimethylcyclohex-1-enyl)methanol

A solution of 7-bromoisoquinoline (958 mg, 4.6 mmol) in tetrahydrofuran(15 mL) was added dropwise into n-butyl lithium (1.6 M in hexane, 2.87mL, 4.6 mmol) at −78° C. and stirred at −78 ° C. for 30 minutes. Then2,6,6-trimethylcyclohex-1-enecarbaldehyde (350 mg, 2.3 mmol) intetrahydrofuran (2 mL) was added and the reaction was stirred at −78° C.for 1 hour and then allowed to warm to room temperature. The reactionwas quenched with saturated aqueous ammonium chloride and the organicswere extracted with ethyl acetate. The organic phase was washed withsaturated aqueous sodium bicarbonate and brine, dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to give the title compound(794 mg, Yield: 100%). R_(f) 0.5 (2:1 petroleum ether/ethyl acetate); ¹HNMR (400 MHz, CDCl₃) δ 9.24 (s, 1 H), 8.49 (d, J=5.6 Hz, 1 H), 8.04 (s,1 H), 7.78 (s, 2 H), 7.64 (d, J=5.6 Hz, 1 H), 5.56 (s, 1 H), 2.05-2.00(m, 2 H), 1.70-1.56 (m, 4 H), 1.35 (s, 3 H), 1.24 (s, 3 H), 1.16 (s, 3H) ppm; Mass spectrum (ESI+ve) m/tz 282 (M+H⁺).

Example 6b isoquinolin-7-yl(2,6,6-trimethylcyclohex-1-enyl)methanone

To the product of Example 6a in dichloromethane (15 mL) at 0° C. wasadded Dess-Martin periodinane (1.8 g, 4.2 mmol) and the reaction wasstirred at for 1 hour. The mixture was diluted with petroleum ether andfiltered. The filtrate was concentrated under reduced pressure and theresidue purified by silica gel column chromatography to give the titlecompound (610 mg, Yield: 77%). R_(f) 0.4 (5:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 9.40 (s, 1 H), 8.64 (d, J =6.0 Hz, 1H), 8.53 (s, 1 H), 8.29 (dd, J₁=8.8 Hz, J₂=1.6 Hz, 1 H), 7.91 (d, J=8.4Hz, 1 H), 7.72 (d, J=6.0 Hz, 1 H), 2.16 (t, J=6.4 Hz, 2 H), 1.85-1.82(m, 2 H), 1.63-1.61 (m, 2 H), 1.48 (s, 3 H), 1.08 (s, 6 H) ppm.

Example 6c(1,2,3₁4-tetrahydroisoquinolin-7-yl)(2,6,6-trimethylcyclohex-1-enyl)methanone

To the product of Example 6b (350 mg, 1.25 mmol) in acetic acid (2 mL)was added platinum dioxide (35 mg) and the reaction was stirred under anatmosphere of hydrogen for 4 hours at room temperature. The mixture wasfiltered and the filtrate concentrated under reduced pressure. Theresidue was dissolved in ethyl acetate and adjusted to pH=8 withsaturated aqueous sodium bicarbonate. The aqueous layer was extractedwith ethyl acetate and the organic layer was washed with brine, driedover anhydrous sodium sulfate and concentrated under reduced pressure.The residue was purified by silica gel column chromatography to give thetitle compound (206 mg, Yield: 58%). R_(f) 0.5 (10:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.67 (d, J=7.6 Hz,1 H), 7.62 (s, 1 H), 7.16 (d, J=8.0 Hz, 1 H), 4.08 (s, 2 H), 3.18 (t,J=6.0 Hz, 2 H), 2.87 (t, J=6.0 Hz, 2 H), 2.07 (t, J=6.4 Hz, 2 H),1.78-1.74 (m, 2 H), 1.53-1.50 (m, 2 H), 1.43 (s, 3 H), 1.02 (s, 6 H)ppm.

Example 67-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 6c (204 mg, 0.72 mmol) indichloromethane (10 mL) was added triethylamine (291 mg, 2.88 mmol) andisocyanatotrimethylsilane (249 mg, 2.16 mmol) and the reaction mixturewas stirred at room temperature overnight after which additionaltriethylamine (291 mg, 2.88 mmol) and isocyanatotrimethylsilane (249 mg,2.16 mmol) were added and stirring was continued for 5 hours. Themixture was diluted with dichloromethane and washed with water,saturated aqueous ammonium chloride, saturated aqueous sodiumbicarbonate and brine. The organic phase was dried over anhydrous sodiumsulfate and then concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to afford the titlecompound as a white solid (160 mg, Yield: 68%). Mp=92.5-93.6° C.; R_(f)0.4 (15:1 dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.75 (d,J=8.0 Hz, 1 H), 7.73 (s, 1 H), 7.24 (d, J=8.0 Hz, 1 H), 4.63 (s, 2 H),4.55 (bs, 2 H), 3.66 (t, J=6.0 Hz, 2 H), 2.95 (t, J=5.6 Hz, 2 H), 2.08(t, J=6.4 Hz, 2 H), 1.79-1.76 (m, 2 H), 1.56-1.54 (m, 2 H), 1.43 (s, 3H), 1.02 (s, 6 H) ppm; Mass spectrum (ESI+ve) m/z 327 (M+H⁺).

Example 77-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 6 (73.4 mg, 0.22 mmol) intetrahydrofuran (2 mL) at 0° C. was added lithium aluminum hydride (51mg, 1.35 mmol) and the reaction was stirred at 0° C. for 2 hours. Themixture was quenched with saturated aqueous ammonium chloride and theorganics were extracted with ethyl acetate. The combined organic phasewas washed with saturated aqueous sodium bicarbonate, brine, dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by prep-TLC to afford the title compound as a whitesolid (44 mg, Yield: 59%). Mp=88.2-89.5° C.; R_(f) 0.3 (15:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.24-7.20 (m, 2 H),7.10 (d, J=7.6 Hz, 1 H), 5.37 (s, 1 H), 4.56 (s, 2 H), 4.54 (s, 2 H),3.63 (t, J=6.0 Hz, 2 H), 2.88 (t, J=5.8 Hz, 2 H), 1.99 (t, J=6.0 Hz, 2H), 1.82 (s, 1 H), 1.68-1.64 (m, 2 H), 1.56-1.52 (m, 2 H), 1.39 (s, 3H), 1.18 (s, 3 H), 1.04 (s, 3 H) ppm; Mass spectrum (ESI+ve) m/z 311(M−H₂O+H⁺).

Example 86-(2,5,5-trimethylcyclopent-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamideExample 8a ethyl 1-methyl-2-oxocyclopentanecarboxylate

To a stirred suspension of dry potassium carbonate (16.64 g, 120.4 mmol)in dry acetone (200 mL) under argon was added ethyl2-oxocyclopentanecarboxylate (9.40 g, 60.2 mmol) followed by methyliodide (7.5 mL, 120.4 mmol). The reaction was heated to reflux for 3hours and then another portion of methyl iodide (7.5 mL, 120.4 mmol) wasadded and the reaction was refluxed overnight. The reaction mixture wascooled and then filtered through a silica gel pad and the solid waswashed with acetone (100 mL×3). The organic phase was concentrated underreduced pressure and the residue dispersed in acetone (200 mL) andfiltered. The filtrate was concentrated under reduced pressure and theresidue was purified by silica gel column chromatography (eluent:petroleum ether/ethyl acetate=100/1→50/1) to afford the title compoundas a colorless liquid (8.35 g, Yield: 82%). ¹H NMR (400 MHz, CDCl₃) δ4.22-4.10 (m, 2H), 2.57-2.40 (m, 2H), 2.36-2.27 (m, 1H), 2.11-2.00 (m,1H), 1.98-1.83 (m, 2H), 1.31 (s, 3H), 1.25 (t, J=7.0 Hz, 3H) ppm.

Example 8b ethyl 1,3,3-trimethyl-2-oxocyclopentanecarboxylate

To a solution of potassium tert-butoxide (16.53 g, 147.3 mmol) inanhydrous tetrahydrofuran (200 mL) at −60° C. under argon was added asolution of the product of Example 8a (8.35 g, 49.1 mmol) in anhydroustetrahydrofuran (20 mL) dropwise during 15 minutes. The reaction mixturewas allowed to warm gradually to −30° C. over 2 hours, after which timethe reaction was cooled down to −60° C. and methyl iodide (18.4 mL,294.6 mmol) was added dropwise. The reaction was then allowed to warmgradually to room temperature and stirred overnight. The mixture waspoured into cooled saturated aqueous ammonium chloride (600 mL) and thenthe organics were extracted with diethyl ether (250 mL×4). The combinedorganic phase was washed with brine (600 mL), dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (eluent: petroleumether/ethyl acetate=50/1) to afford the title compound as a light yellowliquid (7.22 g, Yield: 74%) ¹H NMR (400 MHz, CDCl₃) δ 4.20-4.09 (m, 2H),2.49-2.41 (m, 1H), 1.98-1.88 (m, 1H), 1.86-1.77 (m, 2H), 1.32 (s, 3H),1.24 (t, J=7.0 Hz, 3H), 1.15 (s, 3H), 1.09 (s, 3H) ppm.

Example 8c potassium 1,3,3-trimethyl-2-oxocyclopentanecarboxylate

To a stirred solution of the product of Example 8b (6.72 g, 33.9 mmol)in methanol (34 mL) was added a cooled aqueous potassium hydroxidesolution (1.0 M, 68 mL, 68 mmol). The reaction was then stirred at roomtemperature overnight. The material was used directly for the next step.

Example 8d 2,2,5-trimethylcyclopentanone

A solution of the product of Example 8c in methanol and water (7.4 g,−35.7 mmol) was acidified with concentrated hydrochloric acid to pH=1and then heated to reflux for 1 hour. The reaction mixture was cooledand then diluted with water (70 mL) and the organics were extracted withdiethyl ether (80 mL×4). The combined organic phase was washed withbrine (160 mL), dried over anhydrous sodium sulfate and concentratedunder reduced pressure to give the title compound as a light yellowliquid contaminated with some solvent (4.93 g, Purity: ˜85%). ¹H NMR(400 MHz, CDCl₃) δ 2.26-2.09 (m, 2H), 1.85-1.79 (m, 1H), 1.72-1.64 (m,1H), 1.54-1.45 (m, 1H), 1.12 (d, J=6.8 Hz, 3H), 1.08 (s, 3H), 0.98 (s,3H) ppm.

Example 8e 2,2,5-trimethyl-1-trimethylsilyloxy)cyclopentanecarbonitrile

To a solution of the product of Example 8d (4.93 g, ˜85% purity, ˜33.2mmol) in dichloromethane (120 mL) at room temperature was added zinciodide (265 mg, 0.83 mmol) followed by trimethylsilanecarbonitrile (4.29g, 43.2 mmol). The reaction was stirred at room temperature for 4 hours.The reaction was concentrated under reduced pressure and the resultingmaterial was dispersed in light petroleum ether (250 mL). The mixturewas then filtered through a silica gel pad and the filtrate wasconcentrated under reduced pressure to give the title compound ascolorless a liquid (6.33 g, Yield: 84%, Diastereomer ratio: 4:1). ¹H NMR(400 MHz, CDCl₃) (Major isomer) δ 2.30-2.20 (m, 1H), 1.94-1.82 (m, 1H),1.72-1.50 (m, 2H), 1.38-1.26 (m, 1H), 1.18 (d, J=6.8 Hz, 3H), 1.17 (s,3H), 0.96 (s, 3H), 0.24 (s, 9H) ppm.

Example 8f 1-hydroxy-2,2,5-trimethylcyclopentanecarbonitrile

To a stirred solution of the product of Example 8e (6.33 g, 28.1 mmol)in tetrahydrofuran (56 mL) at room temperature was added 10% aqueoushydrochloric acid (84 mL). The reaction was heated to 45° C. for 4hours. The reaction mixture was concentrated under reduced pressure toremove the tetrahydrofu ran and the aqueous residue was extracted withethyl acetate (80 mL×4). The combined organic phase was washed withbrine (160 mL), dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=100/1->5/1) toafford the title compound as a white solid as a mixture of diastereomers(3.82 g, Yield: 89%). ¹H NMR (400 MHz, CDCl₃) (Major isomer) δ 2.34 (s,1H), 2.33-2.24 (m, 1H), 2.01-1.92 (m, 1H), 1.72-1.59 (m, 2H), 1.41-1.31(m, 1H), 1.24 (d, J=6.8 Hz, 3H), 1.23 (s, 3H), 1.04 (s, 3H) ppm; ¹H NMR(400 MHz, CDCl₃) (Minor isomer) δ 2.61-2.51 (m, 1H), 2.29 (s, 1H),2.01-1.91 (m, 1H), 1.80-1.72 (m, 1H), 1.58-1.51 (m, 1H), 1.47-1.38 (m,1H), 1.16-1.14 (m, 9H) ppm.

Example 8g 2,5,5-trimethylcyclopent-1-enecarbonitrile

A solution of the product of Example 8f (1.54 g, 10.0 mmol) in thionylchloride (6 mL) was heated to 84° C. overnight in a sealed vessel. Thereaction mixture was cooled and then poured into ice-water (100 mL). Theorganics were extracted with diethyl ether (80 mL×4). The combinedorganic phase was washed with saturated aqueous sodium bicarbonate (160mL×2) and brine (160 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to give the title compound as abrown oil (1.20 g,

Yield: 89%). ¹ H NMR (400 MHz, CDCl₃) δ 2.45 (t, J=7.4 Hz, 2H), 1.96 (s,3H), 1.80 (t, J=7.4 Hz, 2H), 1.17 (s, 6H) ppm.

Example 8h 2,5,5-trimethylcyclopent-1-enecarbaldehyde

To a stirred solution of the product of Example 8g (600 mg, 4.44 mmol)in anhydrous dichloromethane (30 mL) at −78° C. under argon was addeddiisobutyl aluminum hydride (1.0 M in hexane, 8.9 mL, 8.9 mmol). Thereaction was kept at −78° C. for 2 hours. The mixture was diluted withdiethyl ether (150 mL) and quenched by addition of wet sodium sulfate.The mixture was then stirred for 30 minutes, filtered and the filtrateconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (eluent: petroleum ether/ethylacetate=50/1→20/1) to afford the title compound as a light yellow liquid(420 mg, Yield: 68%). ¹H NMR (400 MHz, CDCl₃) δ 9.98 (s, 1H), 2.45 (t,J=7.4 Hz, 2H), 2.10 (s, 3H), 1.67 (t, J=7.4 Hz, 2H), 1.22 (s, 6H) ppm.

Example 8i isoquinolin-6-yl(2,5,5-trimethylcyclopent-1-enyl)methanol

A solution of 6-bromoisoquinoline (603 mg, 2.9 mmol) in tetrahydrofuran(10 mL) was added dropwise into n-butyl lithium (1.6 M in hexane, 1.8mL, 2.9 mmol) at −78° C. and stirred at −78° C. for 1 hour. Then theproduct of 8h (200 mg, 1.45 mmol) in tetrahydrofuran (2 mL) was addedand stirred at −78° C. for 1 hour after which time the reaction wasslowly warmed to room temperature. The mixture was quenched withsaturated aqueous ammonium chloride and the organics were extracted withethyl acetate. The organic phase was washed with saturated aqueoussodium bicarbonate, brine, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to give the title compound (344 mg, Yield:89%). R_(f) 0.5 (2:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz,CDCl₃) δ 9.22 (s, 1 H), 8.51 (d, J=5.6 Hz, 1 H), 7.98 (s, 1 H), 7.90 (d,J=8.8 Hz, 1 H), 7.67 (d, J=6.0 Hz, 1 H), 7.54 (d, J=8.4 Hz, 1 H), 5.65(s, 1 H), 2.30 (t, J=7.2 Hz, 2 H), 1.69 (t, J 32 6.8 Hz, 2 H), 1.63 (s,3 H), 1.17 (s, 3 H), 0.93 (s, 3 H) ppm.

Example 8j isoquinolin-6-yl(2,5,5-trimethylcyclopent-1-enyl)methanone

To the product of Example 8i in dichloromethane (20 mL) at 0° C. wasadded Dess-Martin periodinane (818 mg, 1.92 mmol) and the reaction wasstirred at 0° C. for 2 hours. Petroleum ether was added to the mixtureand then it was filtered. The filtrate was concentrated under reducedpressure and the residue was purified by silica gel columnchromatography to give the title compound (240 mg, Yield: 70%). R_(f)0.7 (2:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 9.36(s, 1 H), 8.63 (d, J=5.6 Hz, 1 H), 8.23 (s, 1 H), 8.03 (dd, J₁=8.4 Hz,J₂=1.2 Hz, 2 H), 7.80 (d, J=6.0 Hz, 1 H), 2.50 (t, J=7.2 Hz, 2 H), 1.86(t, J=7.2 Hz, 2 H), 1.48 (s, 3 H), 1.28 (s, 6 H) ppm.

Example 8k(1,2,3,4-tetrahydroisoquinolin-6-yl)(2,5,5-trimethylcyclopent-1-enyl)methanone

To the product of Example 8j (240 mg, 0.94 mmol) in acetic acid (5 mL)was added platinum dioxide (50 mg) and the reaction was stirred under anatmosphere of hydrogen at room temperature for 4 hours. The mixture wasfiltered and the filtrate was concentrated under reduced pressure. Theresidue was dissolved in ethyl acetate and adjusted to pH=8 withsaturated aqueous sodium bicarbonate. The aqueous layer was extractedwith ethyl acetate and the combined organic phase was washed with brine,dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (108 mg, Yield: 43%). R_(f) 0.5 (10:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.58 (s, 1 H), 7.57(d, J=6.4 Hz, 1 H), 7.07 (d, J=8.0 Hz, 1 H), 4.06 (s, 2 H), 3.16 (t,J=6.0 Hz, 2 H), 2.85 (t, J=6.0 Hz, 2 H), 2.43 (t, J=6.8 Hz, 2 H), 1.80(t, J=7.6 Hz, 2 H), 1.50 (s, 3 H), 1.21 (s, 6 H) ppm.

Example 86-(2,5,5-trimethylcyclopent-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 8k (108 mg, 0.4 mmol) indichloromethane (10 mL) was added triethylamine (162 mg, 1.6 mmol) andisocyanatotrimethylsilane (138 mg, 1.2 mmol) and the reaction mixturewas stirred at room temperature overnight. The mixture was diluted withdichloromethane and the organic phase was washed with water, saturatedaqueous ammonium chloride, saturated aqueous sodium bicarbonate andbrine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound as a white solid (104 mg,Yield: 83%). Mp=40.1-41.4° C.; R_(f) 0.3 (15:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.64 (d, J=4.4 Hz,2 H), 7.20 (d, J=8.4 Hz, 1 H), 4.64 (s, 2 H), 4.57 (s, 2 H), 3.64 (t,J=6.0 Hz, 2 H), 2.96 (t, J=6.0 Hz, 2 H), 2.44 (t, J=6.8 Hz, 2 H), 1.80(t, J=7.2 Hz, 2 H), 1.50 (s, 3 H), 1.21 (s, 6 H) ppm; Mass spectrum(ESI+ve) m/z 313 (M+H⁺).

Example 96-(hydroxy(2,5,5-trimethylcyclopent-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1 H)-carboxamide

To a solution of the product of Example 8 (49 mg, 0.16 mmol) intetrahydrofuran (2 mL) at 0° C. was added lithium aluminum hydride (36mg, 0.94 mmol) and the reaction was stirred at 0° C. for 2 hours. Themixture was quenched with wet sodium sulfate and then the reactionmixture was filtered. The filtrate was concentrated under reducedpressure and the residue was dissolved in ethyl acetate. The organicphase was washed with saturated aqueous ammonium chloride, saturatedaqueous sodium bicarbonate, brine, dried over anhydrous sodium sulfateand concentrated under reduced pressure. The residue was purified byprep-TLC to afford the title compound as a white solid (27 mg, Yield:55%). Mp=68.6-69.6° C.; R_(f) 0.5 (10:1)dichloromethane/methanol); ¹HNMR (400 MHz, CDCl₃) δ 7.24 (s, 1 H), 7.21 (d, J=8.0 Hz, 1 H), 7.07 (d,J=8.0 Hz, 1 H), 5.42 (d, J=2.8 Hz, 1 H), 4.56 (s, 2 H), 4.51 (s, 2 H),3.63 (t, J=5.6 Hz, 2 H), 2.89 (t, J=6.0 Hz, 2 H), 2.28 (t, J=7.2 Hz, 2H), 1.72 (d, J=4.0 Hz, 1 H), 1.68-1.64 (m, 5 H), 1.06 (s, 3 H), 0.97 (s,3 H) ppm; Mass spectrum (ESI+ve) m/z 315 (M+H⁺).

Example 106-(3,3,6,6-tetramethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide Example 10a 1,4,4-trimethylcyclohex-2-enol

To a stirred solution of 4,4-dimethylcyclohex-2-enone (40.0 g, 322 mmol)in anhydrous diethyl ether (400 mL) at −78° C. was added an etherealsolution of methyllithium (220 mL of a 1.6 M). The resulting solutionwas allowed to warm to room temperature, stirred for 18 hours. Thereaction was quenched by the addition of water (200 mL). The phases wereseparated and the aqueous layer extracted with diethyl ether ether(2×200 mL). The combined organic phase was washed with water (2×200 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to give the title compound as clear, light yellow oil (41 g,Yield: 90%). R_(f) 0.5 (5:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 5.46 (d, J=10.0 Hz, 1H), 5.43 (d, J=10.0 Hz, 1H),1.73-1.70 (m, 2H), 1.59-1.56 (m, 1H), 1.50-1.45 (m, 1H), 1.27 (s, 3H),1.01 (s, 3H), 0.95 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 123(M-H₂O+H⁺).

Example 10b 3,6,6-trimethylcyclohex-2-enone

To a stirred slurry of pyridinium chlorochromate (123 g, 570 mmol), indichloromethane (840 mL), at room temperature was added in one portion asolution of the product of Example 10a (40.0 g, 285 mmol) indichloromethane (280 mL). The resulting dark red mixture was allowed tostir for 18 hours after which it was filtered and the precipitate washedwith diethyl ether (200 mL). The filtrate was washed successively with5% aqueous sodium hydroxide (2×200 mL), 5% aqueous hydrochloric acid(200 mL), saturated aqueous sodium bicarbonate (2×50 mL), and dried overanhydrous magnesium sulfate, filtered and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto afford the title compound as a colorless oil (14 g, Yield: 35%).R_(f) 0.4 (5:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ5.77 (s, 1H), 2.29 (t, J=6.0 Hz, 2H), 1.93 (s, 3H), 1.80 (d, J=6.0 Hz,3H), 1.09 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 139 (M+H⁺).

Example 10c 2,2,5,5-tetramethylcyclohexanone

Cuprous iodide (6.9 g, 36.2 mmol) was added to a dry 250-mL round-bottomflask equipped with a stir bar and sealed under argon with a septum. Theflask was evacuated with a vacuum pump and purged with argon. Thisprocess was repeated three times. Anhydrous tetrahydrofuran (75 mL) wasinjected into the flask and the slurry was cooled to −78° C. at whichtime methly lithium (45 mL, 72 mmol) was added dropwise. The mixture wasallowed to warm until becoming homogeneous and then it was recooled to−78° C. and boron trifluoride etherate (8.9 mL, 72 mmol) was added via asyringe. The product of Example 10b (5.0 g, 36.2 mmol) was added neatand the reaction mixture was stirred for 1.5 hours. The reaction wasquenched with a solution of 10% aqueous ammonium hydroxide/90% aqueousammonium chloride (250 mL). The organics were extracted with ethylacetate (250 mL) and the organic layer was washed with saturated aqueoussodium bicarbonate (50 mL×2), brine (50 mL), dried over anhydrous sodiumsulfate and concentrated to give 3.5 g a colorless oil which waspurified by silica gel column chromatography to afford the titlecompound as a colorless solid (1.5 g, Yield: 26%). ¹H NMR (400 MHz,CDCl₃) δ 2.21 (s, 2H), 1.69-1.65 (m, 2H), 1.61-1.57 (m, 2H), 1.09 (s,6H), 0.94 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 216.36, 51.32, 44.00,36.89, 36.62, 34.69, 28.5, 25.15 ppm; Mass spectrum (ESI+ve) m/z 155(M+H⁺).

Example 10d2,2,5,5-tetramethyl-1-(trimethylsilyloxy)cyclohexanecarbonitrile

To a mixture of the product of Example 10c (2.7 g, 17.5 mol) indichloromethane (100 mL) was added zinc iodide (140 mg, 0.44 mmol) andtrimethylsilanecarbonitrile (2.27 g, 22.8 mmol) and the reaction wasstirred at room temperature overnight. The organic phase was washed withwater (20 mL) and brine (20 mL) dried over anhydrous sodium sulfate andconcentrated under reduced pressure to give the title compound as ayellow oil (3.8 g, Yield: 86%). ¹H NMR (400 MHz, CDCl₃) δ0 1.81-1.65 (m,3H), 1.36-1.30 (m, 3H), 1.11 (s, 3H), 1.00 (s, 6H), 0.98 (s, 3H), 0.24(s, 9H) ppm.

Example 10e 1-hydroxy-2,2,5,5-tetramethylcyclohexanecarbonitrile

To a mixture of the product of Example 10d (3.8 g, 15.0 mol) intetrahydrofuran (29 mL) was added 10% hydrochloric acid (75 mL) and thereaction was stirred at 44° C. for 5 hours. The reaction wasconcentrated under reduced pressure and the residue diluted with diethylether. The organic phase was washed with water (20 mL) and brine (20mL), dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the the title compound as a colorless oil (2.22 g, Yield: 82%).¹H NMR (400 MHz, CDCl₃) δ 2.21 (s, 1H), 1.87 (d, J=14.4 Hz, 1H), 1.72(d, J=14.4 Hz, 1H), 1.66-1.60 (m, 1H), 1.42-1.34 (m, 3H), 1.17 (s, 3H),1.06 (s, 3H), 1.05 (s, 3H), 1.04 (s, 3H) ppm.

Example 10f 3,3,6,6-tetranmethylcyclohex-1-enecarbonitrile

To a mixture of the product of Example 10e (2.22 g, 12.3 mmol) inpyridine (40 mL) was added thionyl chloride (4.4 mL, 61.3 mmol) and thereaction was stirred at room temperature for 2 hours. The reaction wasacidified with 5 N hydrochloric acid to pH=1, then it was poured intoice-water (20 mL). The aqueous mixture was extracted with ethyl acetate(20 ml×3) and the organic phase washed with water (20 mL), brine (20mL), dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto afford the title compound as a colorless oil (600 mg, Yield: 30%). ¹HNMR (400 MHz, CDCl₃) δ 6.23 (s, 1H), 1.56 (t, J=2.8 Hz, 2H), 1.56-1.43(m, 4H), 1.16 (s, 6H), 1.03 (s, 6H) ppm.

Example 10g 3,3,6,6-tetramethylcyclohex-1-enecarbaldehyde

To the mixture of the product of Example 10f (500 mg, 3.07 mmol) in drydichloromethane (12.5 mL) at −78° C. was added diisobutyl aluminumhydride (6.5 mL, 6.14 mmol) and the reaction was warmed to roomtemperature and stirred for approximately 2.5 hours. Wet sodium sulfatewas added to quench the reaction. The reaction mixture was filtered andthe filtrate was concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to afford the titlecompound as a colorless oil. (360 mg, Yield: 71%). ¹H NMR (400 MHz,CDCl₃) δ 9.31 (s, 1H), 6.33 (s, 1H), 1.55-1.49 (m, 4H), 1.20 (s, 6H),1.10 (s, 6H) ppm.

Example 10h isoquinolin-6-03,3,6,6-tetramethylcyclohex-1-enyl)methanol

A solution of 6-bromoisoquinoline (416 mg, 2 mmol) in tetrahydrofuran (8mL) was added dropwise into n-butyl lithium (1.6 M in hexane, 1.25 mL, 2mmol) at −78° C. and the reaction was stirred for 30 minutes. A solutionof the product of Example 10g (150 mg, 1 mmol) in tetrahydrofuran (2 mL)was added and the reaction was stirred at −78° C. for 1 hour and thenslowly warmed to room temperature. The mixture was quenched with theaddition of saturated aqueous ammonium chloride and the mixture wasextracted with ethyl acetate. The organic phase was washed withsaturated aqueous sodium bicarbonate, brine, dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to afford the titlecompound (167.3 mg, Yield: 63%). ¹H NMR (400 MHz, CDCl₃) δ 9.22 (s, 1H),8.52 (d, J=6.0 Hz, 2H), 7.96 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.4 Hz, 1H),5.49 (s, 1H), 5.32 (d, J=15.2 Hz, 1H), 1.63-1.44 (m, 4H), 1.21 (s, 3H),1.01 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H) ppm.

Example 10iisoquinolin-6-yl(3,3,6,6-tetramethylcyclohex-1-enyl)methanone

To the product of Example 10h (167.3 mg, 0.57 mmol) in dichloromethane(10 mL) at room temperature was added sodium bicarbonate (50 mg) andDess-Martin periodinane (365 mg, 0.85 mmol) and the reaction was stirredat room temperature overnight. The mixture was concentrated underreduced pressure and the residue was purified by silica gel columnchromatography to afford the title compound as a colorless oil (130 mg,Yield: 78%). ¹H NMR (400 MHz, CDCl₃) δ 9.32 (s, 1H), 8.61 (d, J=6.0 Hz,1H), 8.09 (s, 1H), 8.02 (d, J=8.4 Hz, 1H), 7.91 (d, J=8.4 Hz, 1H), 7.73(d, J=6.0 Hz, 1H), 5.91 (s, 1H), 1.66-1.59 (m, 4H), 1.31 (s, 6H), 1.07(s, 6H) ppm.

Example 10j(1,2,3,4-tetrahydroisoquinolin-6-yl)(3,3,6,6-tetramethylcyclohex-1-enyl)methanone

To the product of Example 10i (130 mg, 0.47 mmol) in acetic acid (2 mL)was added platinum dioxide (20 mg) and the reaction mixture was stirredunder an atmosphere of hydrogen for 3 hours. The mixture was filteredand the filtrate was concentrated under reduced pressure. The residuewas diluted with saturated aqueous sodium bicarbonate and the aqueouslayer was extracted with ethyl acetate. The organic phase was washedwith brine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to afford the title compound (59 mg, Yield: 42%). ¹H NMR(400 MHz, CDCl₃) δ 7.50 (s, 1H), 7.49 (d, J=8.0 Hz, 1H), 7.06 (d, J=8.0Hz, 1H), 5.77 (s, 1H), 4.11 (s, 2H), 3.21 (t, J=5.6 Hz, 2H), 2.90 (t,J=5.6 Hz, 2H), 2.32 (s, 1H), 1.58 (s, 4H), 1.24 (s, 6H), 1.05 (s, 6H)ppm.

Example 106-(3,3,6,6-tetramethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 10j (59 mg, 0.2 mmol) indichloromethane (5 mL) was added triethylamine (110 mg, 1.0 mmol) andisocyanatotrimethylsilane (94 mg, 0.8 mmol) and stirred at roomtemperature overnight. The mixture was diluted with dichloromethane andwashed with water, saturated aqueous ammonium chloride, saturatedaqueous sodium bicarbonate and brine, dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to afford the titlecompound as a white solid (47 mg, Yield:

69%). Mp=78.6-79.7° C.; R_(f) 0.3 (10:1 dichloromethane/methanol); ¹HNMR (400 MHz, CDCl₃) δ 7.56 (s, 1H), 7.53 (d, J=8.0 Hz, 2H), 7.17 (d,J=7.6 Hz, 1H), 5.79 (s, 1H), 4.63 (s, 2H), 4.52 (s, 2H), 3.64 (t, J=6.0Hz, 2H), 2.94 (t, J=6.0 Hz, 2H), 1.59 (s, 4H), 1.24 (s, 6H), 1.06 (s,6H) ppm; Mass spectrum (ESI+ve) m/z 341 (M+H⁺).

Example 116-(7,7-dimethylcyclohept-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamideExample 11a (E)-(2,2-dimethylcycloheptylidene)methanol

To a stirred mixture of powdered sodium methoxide (3.0 g, 56.16 mmol) intoluene (90 mL) at 0° C. was added cycloheptanone (6.0 g, 53.49 mmol)and ethyl formate (7.92 g, 106.98 mmol). The mixture was warmed to roomtemperature and stirred overnight. Ice water (70 mL) was added and theorganic phase separated. The organic phase was washed by 5% aqueoussodium hydroxide (30 mL×2) and the combined aqueous phase was acidifiedto pH=3 with 2 N hydrochloric acid and then extracted with ethyl acetate(120 mL×3). The combined organic phase was washed with brine, dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography (eluent:petroleum ether/ethyl acetate =80/1) to afford the title compond as acolorless oil (4.26 g, Yield: 57%). R_(f)=0.6 (20:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 14.68 (d, J=9.2 Hz, 1H), 7.63 (d, J=9.2 Hz, 1 H), 2.55-2.52 (m, 2 H), 2.26-2.24 (m, 2 H),1.76-1.58 (m, 6 H) ppm.

Example 11b (E)-2-(isopropoxymethylene)cycloheptanone

To a suspension of powdered potassium carbonate (6.3 g, 45.48 mmol) inacetone (100 mL) was added the product of Example 11a (4.26 g, 30.19mmol) and 2-iodopropane (3.8 mL, 37.49 mmol). The mixture was refluxedovernight. The reaction was filtered and the filtrate was concentratedunder reduced pressure. The residue was dissolved in ethyl acetate (90mL) and the organic phase was washed with 5% aqueous sodium hydroxide(60 mL×2) and brine, dried over anhydrous sodium sulfate andconcentrated under reduced pressure to give the the title compound as alight yellow oil (5.6 g, Yield: 95%). R_(f)=0.5 (20:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 7.38 (s, 1 H), 4.22-4.16(m, 1 H), 2.56-2.53 (m, 2 H), 2.41-2.39 (m, 2 H), 1.77-1.57 (m, 6 H),1.29 (d, J=6.0 Hz, 6 H) ppm.

Example 11c (E)-7-(isopropoxymethylene)-2,2-dimethytcycloheptanone

To a mixture of potassium tert-butoxide (10.3 g, 92.18 mmol) inanhydrous tetrahydrofuran (140 mL) under argon at 0° C. was added theproduct of Example 11 b (5.6 g, 30.49 mmol) and then methyl iodide (9.56mL, 153.62 mmol). The mixture was stirred for 4.5 hours at roomtemperature. The mixture was filtered and the filtrate concentratedunder reduced pressure and then the residue was dissolved in ethylacetate (60 mL). The organic phase was washed with 5% aqueous sodiumhydroxide (60 mL×2) and brine, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (eluent: petroleum ether/ethyl acetate=70/1)to afford the title compound as a colorless oil (3.82 g, Yield: 59%).R_(f)=0.6 (20:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃)δ 7.15 (s, 1 H), 4.21-4.15 (m, 1 H), 2.91-2.86 (m, 1 H), 2.75-2.70 (m, 1H), 1.98-1.90 (m, 4 H), 1.68-1.54 (m, 2 H), 1.18 (d, J=6.8 Hz, 6 H),1.06 (d, J=6.8 Hz, 3 H).

Example 11d 2,2-dimethylcyclohepta none

To a solution of the product of 11 c (620 mg, 3.44 mmol) in ethanol (6mL) was added a solution of 20% aqueous sodium hydroxide (1.45 g sodiumhydroxide in 6 g of water). The resulting mixture was heated to refluxfor 9 hours. Water (5 mL) was added to the reaction mixture and thereaction was extracted with light petroleum ether. The combined organicphase was washed with brine, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (eluent: petroleum ether/ethyl acetate=60/1)to afford the desired compound as a colorless oil. R_(f)=0.7 (20:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 2.54-2.51 (m,2 H), 1.67-1.61 (m, 4 H), 1.50-1.49 (m, 2 H), 1.33-1.24 (m, 2 H), 1.08(s, 6 H).

Example 11e 2,2-dimethyl-1-(trimethylsilyloxy)cycloheptanecarbonitrile

To a solution of the product of Example 11d (500 mg, 3.57 mmol) indichloromethane (15 ml) at room temperature was added zinc iodide (22.8mg, 0.071 mmol) followed by trimethylsilanecarbonitrile (460.6 g, 4.62mmol). The reaction was stirred at room temperature for 2 hours.Petroleum ether was added and the reaction mixture was filtered and thesolid was washed with additional petroleum ether. The filtrate wasconcentrated under reduced pressure to afford the title compound as ared oil (700 mg, Yield: 82%). R_(f)=0.9 (20:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 1.98 (t, J=5.0 Hz, 2 H), 1.65-1.54(m, 8 H), 1.11 (s, 3 H), 1.01 (s, 3 H), 0.24 (s, 9 H) ppm.

Example 11f 1-hydroxy-2,2-dimethylcycloheptanecarbonitrile

To a solution of the product of Example 11e (700 mg, 2.92 mmol) intetrahydrofuran (8 mL) was added 10% hydrochloric acid (12 mL). Thereaction was stirred at room temperature overnight. The reaction mixturewas then diluted with water (10 ml) and the organics were extracted withethyl acetate (20 mL×3). The combined organic phase was washed withbrine (40 mL), dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=50/1) to affordthe title compound as a colorless oil (430 mg, Yield: 88%). R_(f)=0.3(20:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 2.16 (s,1 H), 2.01-1.98 (m, 2 H), 1.73-1.50 (m, 8 H), 1.17 (s, 3 H), 1.05 (s, 3H) ppm.

Example 11g 7,7-dimethylcyclohept-1-enecarbonitrile

To a mixture of the product of Example 11f (420 mg, 2.51 mmol) inpyridine (17.5 mL) was added thionyl chloride (0.91 mL, 12.56 mmol). Thereaction was stirred at room temperature for 2 hours. The reactionmixture was acidified to pH=1 with 6 N hydrochloric acid and then theorganics were extracted with ethyl acetate (20 mL×3). The combinedorganic phase was washed with brine, dried over anhydrous sodium sulfateand concentrated under reduced pressure. The residue was purified bycolumn chromatography over silica gel (eluent: petroleum ether/ethylacetate=70/1) to afford the title compound as a colorless oil (210 mg,Yield: 56%). R_(f)=0.6 (20:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 6.60 (t, J=6.2 Hz, 2 H), 2.33-2.29 (m, 2 H), 1.78-1.72 (m,2 H), 1.67-1.62 (m, 4 H), 1.23 (s, 6 H) ppm.

Example 11h 7,7-dimethylcyclohept-1-enecarbaldehyde

To a stirred solution of the product of Example 11 g (274 mg, 1.84 mmol)in anhydrous dichloromethane (15 mL) at −78° C. under argon was addeddiisobutyl aluminum hydride (1.0 M in hexane, 3.7 mL, 3.7 mmol) dropwisevia syringe. The reaction was stirred at −78° C. for 2 hours and thendiluted with diethyl ether (30 mL) and quenched by the dropwise additionof tetrahydrofuran/water (3 mL, 5/1). The resulting mixture was warmedto room temperature and stirred for 30 minutes. The reaction wasfiltered and the filtrate was concentrated under reduced pressure. Theresidue was purified by flash column chromatography (eluent: lightpetroleum ether/ethyl acetate=30/1) to afford the title compound as acolorless liquid (214 mg, Purity: ˜83%). R_(f)=0.6 (20:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 9.27 (s, 1 H), 6.63 (t,J=6.0 Hz, 1 H), 2.49-2.44 (m, 2 H), 1.77-1.72 (m, 4 H), 1.67-1.64 (m, 2H), 1.25 (s, 6 H) ppm.

Example 11i (7,7-dimethylcyclohept-1-enyl)(isoquinolin-6-yl)methanol

A solution of 6-bromoisoquinoline (416 mg, 2 mmol) in tetrahydrofuran (8mL) was added dropwise into n-butyl lithium (1.6 M in hexane, 1.25 mL, 2mmol) at −78° C. and stirred at −78° C. for 30 minutes. Then the productof Example 11h (150 mg, 1 mmol) in tetrahydrofuran (2 mL) was added andthe reaction was stirred at −78° C. for 1 hour and then slowly warmed toroom temperature. The mixture was quenched with saturated aqueousammonium chloride and the organics were extracted with ethyl acetate.The organic phase was washed with saturated aqueous sodium bicarbonate,brine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (124 mg, Yield: 45%).R_(f)=0.3 (2:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ9.22 (s, 1 H), 8.52 (d, J=6.0 Hz, 1 H), 7.92 (s, 1 H), 7.90 (d, J=8.8Hz, 1 H), 7.66 (d, J=5.6 Hz, 1 H), 7.50 (d, J=9.2 Hz, 1 H), 5.74 (t,J=6.4 Hz, 1 H), 5.54 (s, 1 H), 2.20-2.15 (m, 2 H), 1.81-1.55 (m, 6 H),1.25 (s, 3 H), 1.14 (s, 3 H) ppm.

Example 11j (7,7-dimethylcyclohept-1-enyl)(isoquinolin-6-yl)methanone

The product of Example 11i in dichloromethane (5 mL) at 0° C. was addedDess-Martin periodinane (280 mg, 0.66 mmol) and the reaction was stirredat 0° C. for 1 hour. The reaction mixture was added to petroleum etherand filtered. The filtrate was concentrated under reduced pressure andthe residue was purified by silica gel column chromatography to give thetitle compound (104 mg, Yield: 85%). R_(f)=0.4 (5:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 9.33 (s, 1 H), 8.62 (d,J=5.6 Hz, 1 H), 8.25 (s, 1 H), 8.08-8.02 (m, 2 H), 7.78 (d, J=6.0 Hz, 1H), 5.99 (t, J=6.0 Hz, 1 H), 2.37-2.33 (m, 2 H), 1.90-1.72 (m, 6 H),1.28 (s, 6 H) ppm.

Example 11k(7,7-dimethylcyclohept-1-enyl)(1,2,3,4-tetrahydroisoquinolin-6-yl)methanone

To the product of Example 11j (104 mg, 0.37 mmol) in acetic acid (2 mL)at room temperature was added platinum dioxide (20 mg) and the reactionwas stirred under an atmosphere of hydrogen for 3 hours. The mixture wasfiltered and the filtrate concentrated under reduced pressure. Theresidue was dissolved in ethyl acetate and adjusted to pH=8 withsaturated aqueous sodium carbonate. The aqueous layer was extracted withethyl acetate. The organic phase was washed with brine, dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to give thetitle compound (79 mg, Yield: 75%). R_(f) 0.5 (10:1dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.58 (s, 1 H), 7.61(s, 1 H), 7.60 (d, J=6.8 Hz, 1 H), 7.06 (d, J=8.0 Hz, 1 H), 5.83 (t,J=6.0 Hz, 1 H), 4.06 (s, 2 H), 3.16 (t, J=6.0 Hz, 2 H), 2.86 (t, J=6.0Hz, 2 H), 2.30 (dd, J₁=12.0 Hz, J₂=6.4 Hz, 2 H), 1.85-1.80 (m, 2 H),1.73-1.69 (m, 4 H), 1.21 (s, 6 H) ppm.

Example 116-(7,7-dimethylcyclohept-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of Example 11k (77 mg, 0.27 mmol) in dichloromethane (5mL) was added triethylamine (110 mg, 1.09 mmol) andisocyanatotrimethylsilane (94 mg, 0.82 mmol) and the reaction wasstirred at room temperature overnight. The mixture was diluted withdichloromethane and the organic phase was washed with water, saturatedaqueous ammonium chloride, saturated aqueous sodium bicarbonate andbrine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by prep-TLC to give the titlecompound as a white solid (67 mg, Yield: 75%). R_(f) 0.6 (10:1)dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.68 (d, J=4.4 Hz,2 H), 7.19 (d, J=8.4 Hz, 1 H), 5.85 (t, J=6.0 Hz, 1 H), 4.63 (s, 2 H),4.56 (s, 2 H), 3.64 (t, J=6.0 Hz, 2 H), 2.96 (t, J=5.6 Hz, 2 H), 2.31(dd, J₁=11.6 Hz, J₂=6.0 Hz, 2 H), 1.85-1.81 (m, 2 H), 1.72-1.70 (m, 4H), 1.22 (s, 6 H) ppm; Mass spectrum (ESI+ve) m/z 327 (M+H⁺).

Example 126-(7,7-dimethylcyclohept-1-enyl)(hydroxy)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of the product of Example 11 (59 mg, 0.18 mmol) intetrahydrofuran (2 mL) at 0° C. was added lithium aluminum hydride (41mg, 1.08 mmol) and the reaction was stirred at 0° C. for 1.5 hours. Themixture was quenched with wet sodium sulfate and then filtered. Thefiltrate was concentrated under reduced pressure and the residue wasdissolved in ethyl acetate and the organic phase was washed withsaturated aqueous ammonium chloride, saturated aqueous sodiumbicarbonate, brine, dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The residue was purified by prep-TLC to give thetitle compound as a white solid (19 mg, Yield: 32%). R_(f) 0.5 (10:1)dichloromethane/methanol; ¹H NMR (400 MHz, CDCl₃) δ 7.19 (d, J=5.2 Hz, 2H), 7.08 (d, J=8.4 Hz, 1 H), 5.87 (t, J=6.4 Hz, 1 H), 5.32 (s, 1 H),4.56 (s, 2 H), 4.53 (s, 2 H), 3.63 (t, J=5.6 Hz, 2 H), 2.89 (t, J=6.0Hz, 2 H), 2.22 (dd, J₁=11.6 Hz, J₂=6.4 Hz, 2 H), 1.77-1.72 (m, 2 H),1.65 (d, J=4.0 Hz, 1 H), 1.61-1.58 (m, 4 H), 1.18 (s, 3 H), 1.00 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 329 (M+H⁺).

Example 136-((R)-1-hydroxy-1-((1R,6S)-2,2,6-trimethylcyclohexyl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxamideExample 13a isoquinolin-6-yl((1R,6S)-2,2,6-trimethylcyclohexyl)methanone

To a solution of the product of Example 3c (200 mg, 0.71 mmol) indichloromethane (8 mL) at 0° C. was added Dess-Martin periodinane (450mg, 1.06 mmol) and the reaction was stirred for 1 hour. The mixture wasquenched with saturated aqueous sodium bicarbonate and the organics wereextracted with ethyl acetate. The organic phase was washed with brine,dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (189 mg, yield: 95%) ¹H NMR (400 MHz, CDCl₃)δ 9.33 (s, 1H), 8.63 (d, J=6.0 Hz, 1H), 8.40 (s, 1H),8.16-8.14 (m, 1H),8.05 (d, J=8.8 Hz, 1H), 7.81 (d, J=5.6 Hz, 1H), 3.17 (d, J=10.8 Hz, 1H),2.11-2.04 (m, 1H), 1.86-1.82 (m, 1H), 1.68-1.40 (m, 4H), 1.36-1.11 (m,1H), 1.02 (s, 3H), 0.81 (d, J=6.8 Hz, 3H), 0.79 (s, 3H).

Example 13b (1,2,3,4-tetrahydroisoquinolin-6-yl)((1R,6S)-2,2,6-trimethylcyclohexyl)methanone

To a solution of the product of Example 13a (178 mg, 0.63 mmol) inacetic acid (2 mL) was added platinum dioxide (25 mg) and the reactionwas stirred at room temperature under an atmosphere of hydrogen for 4hours. The mixture was diluted with dichloromethane (40 mL) and basifiedwith 1 N NaOH (35 mL) and the organic phase was separated. The aqueouslayer was extracted with dichloromethane (30 mL). The combined organicphase was washed with brine (50 mL), dried over anhydrous sodium sulfateand concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (eluent:dichloromethane/methanol=50/1→10/1) to afford the title compound as adark brown oil (130 mg, Yield: 72%). ¹H NMR (400 MHz, CDCl₃) δ 7.76 (d,J=8.4 Hz, 1H), 7.73 (s, 1H), 7.13 (d, J=8.4 Hz, 1H),4.18 (s, 2H), 3.28(t, J=6.2 Hz, 2H), 3.01 (t, J=6.2 Hz, 2H), 2.11-2.02 (m, 1H), 1.81-1.77(m, 1H), 1.61-1.54 (m, 2H), 1.44-1.41 (m, 1H), 1.34-1.24 (m, 3H),1.10-0.98 (m, 4H), 0.77 (s, 3H), 0.74 (d, J=6.4 Hz, 3H) ppm; Massspectrum (ESI+ve) m/z 286 (M+H⁺).

Example 13c(R)-1-(1,2,3,4-tetrahydroisoquinolin-6-yl)-1-((1R,6S)-2,2,6-trimethylcyclohexyl)ethanol

To a stirred solution of the product of Example 13b (95 mg, 0.33 mmol)in anhydrous tetrahydrofuran (6 mL) under argon at −78° C. was addedmethyl lithium (1.6 M in diethyl ether) (1.04 mL, 1.66 mmol). Thereaction was stirred at −78° C. for 1 hour, after which the reaction wasallowed to warm gradually to room temperature and stirring was continuedovernight. The reaction was quenched with saturated aqueous ammoniumchloride (25 mL) and the organics were extracted with ethyl acetate (25mL×3). The combined organic phase was washed with brine (40 ml), driedover anhydrous sodium sulfate and concentrated under reduced pressure.Purification of the residue by silica gel column chromatography (eluent:dichloromethane/methanol=50/1→5/1) afforded 30 mg of the impure titlecompound as a yellow oil which was used directly in the next step. Massspectrum (ESI+ve) m/z 302 (M+H⁺).

Example 136-((R)-1-hydroxy-1-((1R,6S)-2,2,6-trimethylcyclohexyl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To a solution of compound of the crude product of Example 13c (28 mg,0.1 mmol) in dichloromethane (2 mL) was added triethylamine (55 mg, 0.5mmol) and isocyanatotrimethylsilane (47 mg, 0.4 mmol). The reaction wasstirred at room temperature overnight. The mixture was diluted withdichloromethane and washed with water, saturated aqueous ammoniumchloride, saturated aqueous sodium bicarbonate and brine. The organicphase was dried over anhydrous sodium sulfate and concentrated underreduced pressure to afford the title compound as a yellow oil (15 mg).Mass spectrum (LC ESI+ve) m/z 345 (M+H⁺).

Example 146-(hydroxy(3,3,6,6-tetramethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide

To the solution of of the product of Example 10 (6.9 mg, 0.02 mmol) indry tetrahydrofuran (3 mL) at 0° C. was added lithium aluminum hydride(7.8 mg, 0.2 mmol) and the reaction mixture was slowly warmed to roomtemperature and stirred for 1 h. The reaction mixture was quenched withwet sodium sulfate, stirred for 15 minutes and filtered. The filtratewas concentrated under reduced pressure to give the title compound.R_(f) 0.2 (10:1 dichloromethane/methanol); ¹H NMR (400 MHz, CDCl₃) δ7.18 (d, J=8.4 Hz, 2H), 7.07 (d, J=7.6 Hz, 1H), 5.49 (s, 1H), 5.26 (s,1H), 4.56 (s, 2H), 4.50 (s, 2H), 3.62 (t, J=6.0 Hz, 2H), 2.89 (d, J=6.0Hz, 2H), 1.60 (s, 4H), 1.16 (s, 3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.85(s, 3H) ppm; Mass spectrum (ESI+ve) m/z 343 (M+H⁺).

BIOLOGY EXAMPLES

In carrying out the procedures of the present invention it is of courseto be understood that reference to particular buffers, media, reagents,cells, culture conditions and the like are not intended to be limiting,but are to be read so as to include all related materials that one ofordinary skill in the art would recognize as being of interest or valuein the particular context in which that discussion is presented. Forexample, it is often possible to substitute one buffer system or culturemedium for another and still achieve similar, if not identical, results.Those of skill in the art will have sufficient knowledge of such systemsand methodologies so as to be able, without undue experimentation, tomake such substitutions as will optimally serve their purposes in usingthe methods and procedures disclosed herein.

The invention is described in more detail in the following non-limitingexamples. It is to be understood that these particular methods andexamples in no way limit the invention to the embodiments describedherein and that other embodiments and uses will no doubt suggestthemselves to those skilled in the art.

Reagents

Monoclonal anti-rhodopsin 1D4 antibody can be purchased from Universityof British Columbia.

Cell Lines and Culture Conditions

Stable cell lines expressing opsin protein were generated using theFlp-In T-Rex system. The stable cells were grown in DMEM high glucosemedia supplemented with 10% (v/v) fetal bovine serum,antibiotic/antimycotic solution, 5 μ/ml blasticidin and hygromycin at37° C. in presence of 5% CO₂. For all the experiments the cells wereallowed to reach confluence and were induced to produce opsin or amutant opsin with 1 μg/mL tetracycline after change of media and thencompounds were added. The plates were incubated for 48 hours after whichthe cells were harvested.

SDS-PAGE and Western Blotting

Proteins were separated on SDS-PAGE gels and western blotted asdescribed in (Noorwez et al., J. Biol. Chem. 279,16278-16284 (2004)).

The in vivo efficacy of the compounds of the invention in treatingmacular degeneration can be demonstrated by various tests well known inthe art. For example, human patients are selected based on a diagnosisof macular degeneration (such as where there is a gross diagnosis ofthis condition or where they have been shown to exhibit build-up oftoxic visual cycle products, such as A2E, lipofuscin, or drusen in theireyes. A compound of the invention, such as that of Formula I, isadministered to a test group while a placebo, such as PBS or DMSO, isadministered to a control group that may be as large or may be somewhatsmaller than the test group. The test compound is administered either ona one time basis or on a sequential basis (for example, weekly or daily)or according to some other predetermined schedule.

Administration of the test compound is normally by oral or parenteralmeans and in an amount effective to retard the development and/orreoccurrence of macular degeneration. An effective dose amount isgenerally in the range of about 1 to 5,000 mg or in the range of 10 to2,000 mg/kg. Administration may include multiple doses per day.

Efficacy of the test compound in retarding progression of maculardegeneration is generally by measuring increase in visual acuity (forexample, using Early Treatment Diabetic RP Study (ETDRS) charts(Lighthouse, Long Island, N.Y.). Other means of following and evaluatingefficacy is by measuring/monitoring the autofluorescence or absorptionspectra of such indicators as N-retinylidene-phosphatidylethanolamine,dihydro-N-retinylidene-N-retinyl-phosphatidylethanolamine,N-retinylidene-N-retinyl-phosphatidylethanolamine,dihydro-N-retinylidene-N-retinyl-ethanolamine, and/orN-retinylidene-phosphatidylethanolamine in the eye of the patient.Autofluorescence is monitored using different types of instrument, forexample, a confocal scanning laser ophthalmoscope.

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is acommon pathological feature observed in various degenerative diseases ofthe retina. A toxic vitamin A-based fluorophore (A2E) present withinlipofuscin granules has been implicated in death of RPE andphotoreceptor cells. Such experiments can employ an animal model thatmanifests accelerated lipofuscin accumulation to evaluate the efficacyof a therapeutic approach based upon reduction of serum vitamin A(retinol). Administration of test compound to mice harboring a nullmutation in the Stargardt's disease gene (ABCA4) produces reductions inserum retinol/retinol binding protein and arrested accumulation of A2Eand lipofuscin autofluorescence in the RPE.

Test animals are available for use in testing efficacy of a testcompound in reducing build-up of toxic pigments, such as lipofuscin. Forexample, mice have been produced that exhibit increased production ofsuch toxic product. Such mice have been described in the literature(see, for example, Widder et al., U.S. Pub. 2006/0167088) and theirvalue and utility are well known to those in the art.

Showing the efficacy of compounds of the invention in protecting againstlight toxicity is conveniently performed by methods well known in theart (see, for example, Sieving et al, PNAS, Vol. 98. pp 1835-40 (2001)).

Biology Example 1 Rhodopsin Purification and Regeneration

P23H cells were grown to confluency in 10 centermeter plates in DMEMcontaining high glucose, blasticidin (5 μg/ml) and hygromycin (100μg/ml). The cells were induced with tetracycline (1 μg/ml) and treatedwith either DMSO (vehicle) or different concentrations of the testcompound (0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM and 80 μM). After 24 hours,the medium was removed and fresh medium with the the compounds was addedto the plates. β-Ionone (20 μM) was used as a positive control for theexperiments. The cells were harvested 48 hours after the firsttreatment. All procedures from hereon were carried out under a dim redlight (>660 nm). The cells were washed twice with PBS, and incubated for1 hour at room temperature in 1 mL of PBS containing 9-cis-retinal (20μM). After regeneration, the cells were washed with PBS and incubatedfor 1 hour at 4° C. in PBS containing 1% n-dodecyl-β-D maltoside andprotease inhibitors (Roche) for lysis. The cell lysate was centrifugedin a tabletop Beckman ultracentrifuge at 36,000×g for 10 minutes. Thesupernatant was removed and protein was estimated in all of the samples(DC protein assay, Biorad). Equal amounts of protein (5 μg) was loadedon previously prepared 1 D4-coupled cyanogen bromide-activated Sepharose4B beads for 1 hour at 4° C. Briefly, the Sepharose 4B beads wereconjugated with 1D4 antibody that recognizes the C-terminus of opsin.The beads were extensively washed three times with PBS and twice withsodium phosphate buffer (10 mM, pH 6.0), both containing 0.1%n-dodecyl-β-D maltoside. The protein was eluted in the sodium phosphatebuffer containing a synthetic 9 amino acid peptide corresponding to theC-terminus of opsin protein. The eluted rhodopsin was analyzed on aspectrophotometer scanning the UV-visible range from 250 to 650 nm atincrements of 1 nm.

Table 1 contains the results of β-ionone (reference compound 1) and testcompounds in which the 480-500 nm absorbance is expressed as a foldincrease over the DMSO control. FIG. 1 is the spectral results using thereference compound 1 (β-ionone) according to Biology Example 1.

TABLE 1 Fold Increase Concentration Compound Over Control (μM) β-ionone2.4 20 2 2.5 10 3 1.5 10 5 1.9 10Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method of treating an ophthalmic condition in asubject in need thereof, comprising administering to the subject aneffective amount of a compound of Formula I

wherein A is:

R¹ and R² are independently: 1) hydrogen, 2) —CH₃, or 3) —CH₂CH₃; R³is: 1) hydrogen, 2) —CH₃, 3) —CH₂CH₃, or 4) deuteron; R⁴ is: 1)hydrogen, 2) —CH₃, or 3) deuteron; R_(a) and R_(b) are eachindependently: 1) hydrogen, or 2) —CH₃; T is: 1) CH₂, 2) CH₂CH₂, or 3)absent; R_(i) and R_(j) are each independently: 1) hydrogen, 2)hydroxyl, or 3) lower alkyl; R_(i) and R_(j) it, when taken together areoxo (═O); X—Y is: 1) —N(CONH₂)—CH₂—, or 2) —CH₂—N(CONH₂)—; includingpharmaceutically acceptable salts, solvates and hydrates thereof.
 2. Themethod of claim 1, wherein said ophthalmic condition is selected fromthe group consisting of wet or dry age related macular degeneration(ARMD), retinitis pigmentosa (RP), a retinal or macular dystrophy,Stargardt's disease, Sorsby's dystrophy, autosomal dominant drusen,Best's dystrophy, peripherin mutation associate with macular dystrophy,dominant form of Stargart's disease, North Carolina macular dystrophy,light toxicity, normal vision loss related aging and normal loss ofnight vision related to aging.
 3. The method of claim 2, wherein saidophthalmic condition is retinitis pigmentosa (RP).
 4. A method oftreating an ophthalmic condition in a subject in need thereof,comprising administering to the subject an effective amount of acompound selected from the group consisting of:6-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 1);6-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 2);6-((S)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 3);6-((1R,6S)-2,2,6-trimethylcyclohexanecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 4);6-((R)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 5);7-(2,6,6-trimethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 6);7-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 7);6-(2,5,5-trimethylcyclopent-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 8);6-(hydroxy(2,5,5-trimethylcyclopent-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 9);6-(3,3,6,6-tetramethylcyclohex-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 10);6-(7,7-dimethylcyclohept-1-enecarbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 11);6-((7,7-dimethylcyclohept-1-enyl)(hydroxy)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 12);6-((R)-1-hydroxy-1-((1R,6S)-2,2,6-trimethylcyclohexyl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 13); and6-(hydroxy(3,3,6,6-tetramethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 14) including all pharmaceutically acceptable salts, hydrates,or solvates thereof.
 5. The method of claim 1, wherein the compound is6-(hydroxy(2,6,6-trimethylcyclohex-1-enyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 2).
 6. The method of claim 1, wherein the compound is6-((S)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 3).
 7. The method f claim 1, wherein the compound is6-((R)-hydroxy((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxamide(Compound 5).
 8. The method of claim 1, wherein said ophthalmiccondition is related to the formation or accumulation of toxic visualcycle products.
 9. The method of claim 4, wherein said ophthalmiccondition is related to the formation or accumulation of toxic visualcycle products.